Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses

ABSTRACT

Provided are compositions and processes that utilize genomic regions differentially methylated between a mother and her fetus to separate, isolate or enrich fetal nucleic acid from a maternal sample. The compositions and processes described herein are useful for non-invasive prenatal diagnostics, including the detection of chromosomal aneuplodies.

RELATED PATENT APPLICATION

This patent application is a continuation of U.S. patent applicationSer. No. 12/561,241, filed on Sep. 16, 2009, which claims the benefit ofU.S. Provisional Patent Application No. 61/192,264 filed on Sep. 16,2008, entitled PROCESSES AND COMPOSITIONS FOR METHYLATION-BASEDENRICHMENT OF FETAL NUCLEIC ACID FROM A MATERNAL SAMPLE USEFUL FOR NONINVASIVE PRENATAL DIAGNOSES, naming Mathias Ehrich as an inventor, andhaving Attorney Docket No. SEQ-6022-PV. The entire content of theforegoing patent application is incorporated herein by reference,including all text, tables and drawings.

FIELD

Provided in certain embodiments are biomarkers. In some embodiments,biomarkers provided are useful for noninvasive detection of fetalgenetic traits. Certain fetal genetic traits include but are not limitedto presence or absence of fetal nucleic acid.

BACKGROUND

Non-invasive prenatal testing is becoming a field of rapidly growinginterest. Early detection of pregnancy-related conditions, includingcomplications during pregnancy and genetic defects of the fetus is ofcrucial importance, as it allows early medical intervention necessaryfor the safety of both the mother and the fetus. Prenatal diagnosis hasbeen conducted using cells isolated from the fetus through proceduressuch as chorionic villus sampling (CVS) or amniocentesis. However, theseconventional methods are invasive and present an appreciable risk toboth the mother and the fetus. The National Health Service currentlycites a miscarriage rate of between 1 and 2 percent following theinvasive amniocentesis and chorionic villus sampling (CVS) tests.

An alternative to these invasive approaches has been developed forprenatal screening, e.g., to detecting fetal abnormalities, followingthe discovery that circulating cell-free fetal nucleic acid can bedetected in maternal plasma and serum (Lo et al., Lancet 350:485-487,1997; and U.S. Pat. No. 6,258,540). Circulating cell free fetal nucleicacid (cffNA) has several advantages making it more applicable fornon-invasive prenatal testing. For example, cell free nucleic acid ispresent at higher levels than fetal cells and at concentrationssufficient for genetic analysis. Also, cffNA is cleared from thematernal bloodstream within hours after delivery, preventingcontamination from previous pregnancies.

Examples of prenatal tests performed by detecting fetal DNA in maternalplasma or serum include fetal rhesus D (RhD) genotyping (Lo et al., N.Engl. J. Med. 339:1734-1738, 1998), fetal sex determination (Costa etal., N. Engl. J. Med. 346:1502, 2002), and diagnosis of several fetaldisorders (Amicucci et al., Clin. Chem. 46:301-302, 2000; Saito et al.,Lancet 356:1170, 2000; and Chiu et al., Lancet 360:998-1000, 2002). Inaddition, quantitative abnormalities of fetal DNA in maternalplasma/serum have been reported in preeclampsia (Lo et al., Clin. Chem.45:184-188, 1999 and Zhong et al., Am. J. Obstet. Gynecol. 184:414-419,2001), fetal trisomy 21 (Lo et al., Clin. Chem. 45:1747-1751, 1999 andZhong et al., Prenat. Diagn. 20:795-798, 2000) and hyperemesisgravidarum (Sekizawa et al., Clin. Chem. 47:2164-2165, 2001).

SUMMARY

The technology provides inter alia human epigenetic biomarkers that areuseful for the noninvasive detection of fetal genetic traits, including,but not limited to, the presence or absence of fetal nucleic acid, theabsolute or relative amount of fetal nucleic acid, fetal sex, and fetalchromosomal abnormalities such as aneuploidy. The human epigeneticbiomarkers of the technology represent genomic DNA that displaydifferential CpG methylation patterns between the fetus and mother. Thecompositions and processes of the technology allow for the detection andquantification of fetal nucleic acid in a maternal sample based on themethylation status of the nucleic acid in said sample. Morespecifically, the amount of fetal nucleic acid from a maternal samplecan be determined relative to the total amount of nucleic acid present,thereby providing the percentage of fetal nucleic acid in the sample.Further, the amount of fetal nucleic acid can be determined in asequence-specific (or locus-specific) manner and with sufficientsensitivity to allow for accurate chromosomal dosage analysis (forexample, to detect the presence or absence of a fetal aneuploidy).

In an aspect of the technology, a method is provided for enriching fetalnucleic acids from a maternal biological sample, based on differentialmethylation between fetal and maternal nucleic acid comprising the stepsof: (a) binding a target nucleic acid, from a sample, and a controlnucleic acid, from the sample, to a methylation-specific bindingprotein; and (b) eluting the bound nucleic acid based on methylationstatus, wherein differentially methylated nucleic acids elute at leastpartly into separate fractions. In an embodiment, the nucleic acidsequence includes one or more of the polynucleotide sequences of SEQ IDNOs: 1-89. SEQ ID NOs: 1-89 are provided in Table 4. The technologyincludes the sequences of SEQ ID NOs: 1-89, and variations thereto. Inanother embodiment, a control nucleic acid is not included in step (a).

In a related embodiment, a method is provided for enriching fetalnucleic acid from a maternal sample, which comprises the followingsteps: (a) obtaining a biological sample from a woman; (b) separatingfetal and maternal nucleic acid based on the methylation status of aCpG-containing genomic sequence in the sample, wherein the genomicsequence from the fetus and the genomic sequence from the woman aredifferentially methylated, thereby distinguishing the genomic sequencefrom the woman and the genomic sequence from the fetus in the sample. Inanother embodiment, the genomic sequence is at least 15 nucleotides inlength, comprising at least one cytosine, further wherein the region has(1) a genomic locus selected from Table 1; and (2) a DNA sequence of nomore than 10 kb upstream and/or downstream from the locus. Inembodiments, obtaining a biological sample from a woman does not limitthe scope of the technology. This obtaining can refer to actuallycollecting a sample from a woman (e.g., a blood draw) or to receiving asample from elsewhere (e.g., from a clinic or hospital) and performingsteps of a method.

In another related embodiment, a method is provided for enriching fetalnucleic acid from a maternal sample, which comprises the followingsteps: (a) obtaining a biological sample from the woman; (b) digestingor removing maternal nucleic acid based on the methylation status of aCpG-containing genomic sequence in the sample, wherein the genomicsequence from the fetus and the genomic sequence from the woman aredifferentially methylated, thereby enriching for the genomic sequencefrom the fetus in the sample. Maternal nucleic acid may be digestedusing one or more methylation sensitive restriction enzymes thatselectively digest or cleave maternal nucleic acid based on itsmethylation status. In another embodiment, the genomic sequence is atleast 15 nucleotides in length, comprising at least one cytosine,further wherein the region consists of (1) a genomic locus selected fromTable 1; and (2) a DNA sequence of no more than 10 kb upstream and/ordownstream from the locus.

In another aspect of the technology, a method is provided for preparingnucleic acid having a nucleotide sequence of a fetal nucleic acid, whichcomprises the following steps: (a) providing a sample from a pregnantfemale; (b) separating fetal nucleic acid from maternal nucleic acidfrom the sample of the pregnant female according to a differentmethylation state between the fetal nucleic acid and the maternalnucleic acid counterpart, wherein the nucleotide sequence of the fetalnucleic acid comprises one or more CpG sites from one or more of thepolynucleotide sequences of SEQ ID NOs: 1-89 within a polynucleotidesequence from a gene or locus that contains one of the polynucleotidesequences of SEQ ID NOs: 1-89; and (c) preparing nucleic acid comprisinga nucleotide sequence of the fetal nucleic acid by an amplificationprocess in which fetal nucleic acid separated in part (b) is utilized asa template. In another embodiment, a method is provided for preparingnucleic acid having a nucleotide sequence of a fetal nucleic acid, whichcomprises the following steps: (a) providing a sample from a pregnantfemale; (b) digesting or removing maternal nucleic acid from the sampleof the pregnant female according to a different methylation statebetween the fetal nucleic acid and the maternal nucleic acidcounterpart, wherein the nucleotide sequence of the fetal nucleic acidcomprises one or more CpG sites from one or more of the polynucleotidesequences of SEQ ID NOs: 1-89 within a polynucleotide sequence from agene that contains one of the polynucleotide sequences of SEQ ID NOs:1-89; and (c) preparing nucleic acid comprising a nucleotide sequence ofthe fetal nucleic acid. The preparing process of step (c) may be ahybridization process, a capture process, or an amplification process inwhich fetal nucleic acid separated in part (b) is utilized as atemplate. Also, in the above embodiment wherein maternal nucleic acid isdigested, the maternal nucleic acid may be digested using one or moremethylation sensitive restriction enzymes that selectively digest orcleave maternal nucleic acid based on its methylation status. In eitherembodiment, the polynucleotide sequences of SEQ ID NOs: 1-89 may bewithin a polynucleotide sequence from a CpG island that contains one ofthe polynucleotide sequences of SEQ ID NOs: 1-89. The polynucleotidesequences of SEQ ID NOs: 1-89 are further characterized in Tables 1-3herein, including the identification of CpG islands that overlap withthe polynucleotide sequences provided in SEQ ID NOs: 1-89. In anotherembodiment, the nucleic acid prepared by part (c) is in solution. In yetanother embodiment, the method further comprises quantifying the fetalnucleic acid from the amplification process of step (c).

In another aspect of the technology, a method is provided for enrichingfetal nucleic acid from a sample from a pregnant female with respect tomaternal nucleic acid, which comprises the following steps: (a)providing a sample from a pregnant female; and (b) separating orcapturing fetal nucleic acid from maternal nucleic acid from the sampleof the pregnant female according to a different methylation statebetween the fetal nucleic acid and the maternal nucleic acid, whereinthe nucleotide sequence of the fetal nucleic acid comprises one or moreCpG sites from one or more of the polynucleotide sequences of SEQ IDNOs: 1-89 within a polynucleotide sequence from a gene that contains oneof the polynucleotide sequences of SEQ ID NOs: 1-89. In anotherembodiment, the polynucleotide sequences of SEQ ID NOs: 1-89 may bewithin a polynucleotide sequence from a CpG island that contains one ofthe polynucleotide sequences of SEQ ID NOs: 1-89. The polynucleotidesequences of SEQ ID NOs: 1-89 are characterized in Table 1 herein. Inanother embodiment, the nucleic acid separated by part (b) is insolution. In yet another embodiment, the method further comprisesamplifying and/or quantifying the fetal nucleic acid from the separationprocess of step (b).

In another aspect of the technology, a composition is providedcomprising an isolated nucleic acid from a fetus of a pregnant female,wherein the nucleotide sequence of the nucleic acid comprises one ormore of the polynucleotide sequences of SEQ ID NOs: 1-89. In oneembodiment, the nucleotide sequence consists essentially of a nucleotidesequence of a gene, or portion thereof. In another embodiment, thenucleotide sequence consists essentially of a nucleotide sequence of aCpG island, or portion thereof. The polynucleotide sequences of SEQ IDNOs: 1-89 are further characterized in Table 1. In another embodiment,the nucleic acid is in solution. In another embodiment, the nucleic acidfrom the fetus is enriched relative to maternal nucleic acid. In anotherembodiment, the composition further comprises an agent that binds tomethylated nucleotides. For example, the agent may be a methyl-CpGbinding protein (MBD) or fragment thereof.

In another aspect of the technology, a composition is providedcomprising an isolated nucleic acid from a fetus of a pregnant female,wherein the nucleotide sequence of the nucleic acid comprises one ormore CpG sites from one or more of the polynucleotide sequences of SEQID NOs: 1-89 within a polynucleotide sequence from a gene, or portionthereof, that contains one of the polynucleotide sequences of SEQ IDNOs: 1-89. In another embodiment, the nucleotide sequence of the nucleicacid comprises one or more CpG sites from one or more of thepolynucleotide sequences of SEQ ID NOs: 1-89 within a polynucleotidesequence from a CpG island, or portion thereof, that contains one of thepolynucleotide sequences of SEQ ID NOs: 1-89. The polynucleotidesequences of SEQ ID NOs: 1-89 are further characterized in Table 1. Inanother embodiment, the nucleic acid is in solution. In anotherembodiment, the nucleic acid from the fetus is enriched relative tomaternal nucleic acid. Hyper- and hypomethylated nucleic acid sequencesof the technology are identified in Table 1. In another embodiment, thecomposition further comprises an agent that binds to methylatednucleotides. For example, the agent may be a methyl-CpG binding protein(MBD) or fragment thereof.

In some embodiments, a nucleotide sequence of the technology includesthree or more of the CpG sites. In another embodiment, the nucleotidesequence includes five or more of the CpG sites. In another embodiment,the nucleotide sequence is from a gene region that comprises a PRC2domain (see Table 3). In another embodiment, the nucleotide sequence isfrom a gene region involved with development. For example, SOX14— whichis an epigenetic marker of the present technology (See Table 1)—is amember of the SOX (SRY-related HMG-box) family of transcription factorsinvolved in the regulation of embryonic development and in thedetermination of cell fate.

In some embodiments, the genomic sequence from the woman is methylatedand the genomic sequence from the fetus is unmethylated. In otherembodiments, the genomic sequence from the woman is unmethylated and thegenomic sequence from the fetus is methylated. In another embodiment,the genomic sequence from the fetus is hypermethylated relative to thegenomic sequence from the mother. Fetal genomic sequences found to behypermethylated relative to maternal genomic sequence are provided inSEQ ID NOs: 1-59. Alternatively, the genomic sequence from the fetus ishypomethylated relative to the genomic sequence from the mother. Fetalgenomic sequences found to be hypomethylated relative to maternalgenomic sequence are provided in SEQ ID NOs: 60-85. Methylationsensitive restriction enzymes of the technology may be sensitive tohypo- or hyper-methylated nucleic acid.

In another embodiment, the fetal nucleic acid is extracellular nucleicacid. Generally the extracellular fetal nucleic acid is about 500, 400,300, 250, 200 or 150 (or any number there between) nucleotide bases orless. In another embodiment, the digested maternal nucleic acid is lessthan about 90, 100, 110, 120, 130, 140 or 150 base pairs. In a relatedembodiment, the fetal nucleic acid is selectively amplified, captured orseparated from or relative to the digested maternal nucleic acid basedon size. For example, PCR primers may be designed to amplify nucleicacid greater than about 75, 80, 85, 90, 95, 100, 105, 110, 115 or 120(or any number there between) base pairs thereby amplifying fetalnucleic acid and not digested maternal nucleic acid. In anotherembodiment, the nucleic acid is subjected to fragmentation prior tocertain methods of the technology. Examples of methods of fragmentingnucleic acid, include but are not limited to sonication and restrictionenzyme digestion. In some embodiments the fetal nucleic acid is derivedfrom the placenta. In other embodiments the fetal nucleic acid isapoptotic.

In some embodiments, the present technology provides a method in whichthe sample is a member selected from the following: maternal wholeblood, maternal plasma or serum, amniotic fluid, a chorionic villussample, biopsy material from a pre-implantation embryo, fetal nucleatedcells or fetal cellular remnants isolated from maternal blood, maternalurine, maternal saliva, washings of the female reproductive tract and asample obtained by celocentesis or lung lavage. In certain embodiments,the biological sample is maternal blood. In some embodiments, thebiological sample is a chorionic villus sample. In certain embodiments,the maternal sample is enriched for fetal nucleic acid prior to certainmethods of the present technology. Examples of fetal enrichment methodsare provided in PCT Publication Nos. WO/2007140417A2, WO2009/032781A2and US Publication No. 20050164241.

In some embodiments, nucleated and anucleated cell populations areremoved from the sample prior to practicing certain methods of thetechnology (e.g., substantially all nucleated and anucleated cellpopulations are removed). In some embodiments, the sample is collected,stored or transported in a manner known to the person of ordinary skillin the art to minimize degradation or the quality of fetal nucleic acidpresent in the sample.

The sample can be from any animal, including but not limited, human,non-human, mammal, reptile, cattle, cat, dog, goat, swine, pig, monkey,ape, gorilla, bull, cow, bear, horse, sheep, poultry, mouse, rat, fish,dolphin, whale, and shark, or any animal or organism that may have adetectable pregnancy-associated disorder or chromosomal abnormality.

In some embodiments, the sample is treated with a reagent thatdifferentially modifies methylated and unmethylated DNA. For example,the reagent may comprise bisulfite; or the reagent may comprise one ormore enzymes that preferentially cleave methylated DNA; or the reagentmay comprise one or more enzymes that preferentially cleave unmethylatedDNA. Examples of methylation sensitive restriction enzymes include, butare not limited to, HhaI and HpaII.

In one embodiment, the fetal nucleic acid is separated from the maternalnucleic acid by an agent that specifically binds to methylatednucleotides in the fetal nucleic acid. In another embodiment, the fetalnucleic acid is separated or removed from the maternal nucleic acid byan agent that specifically binds to methylated nucleotides in thematernal nucleic acid counterpart. In an embodiment, the agent thatbinds to methylated nucleotides is a methyl-CpG binding protein (MBD) orfragment thereof.

In another aspect of the technology, a method is provided fordetermining the amount or copy number of fetal DNA in a maternal samplethat comprises differentially methylated maternal and fetal DNA. Themethod is performed by a) distinguishing between the maternal and fetalDNA based on differential methylation status; and b) quantifying thefetal DNA of step a). In a specific embodiment, the method comprises a)digesting the maternal DNA in a maternal sample using one or moremethylation sensitive restriction enzymes thereby enriching the fetalDNA; and b) determining the amount of fetal DNA from step a). The amountof fetal DNA can be used inter alia to confirm the presence or absenceof fetal nucleic acid, determine fetal sex, diagnose fetal disease or beused in conjunction with other fetal diagnostic methods to improvesensitivity or specificity. In one embodiment, the method fordetermining the amount of fetal DNA does not require the use of apolymorphic sequence. For example, an allelic ratio is not used toquantify the fetal DNA in step b). In another embodiment, the method fordetermining the amount of fetal DNA does not require the treatment ofDNA with bisulfite to convert cytosine residues to uracil. Bisulfite isknown to degrade DNA, thereby, further reducing the already limitedfetal nucleic acid present in maternal samples. In one embodiment,determining the amount of fetal DNA in step b) is done by introducingone or more competitors at known concentrations. In another embodiment,determining the amount of fetal DNA in step b) is done by RT-PCR, primerextension, sequencing or counting. In a related embodiment, the amountof nucleic acid is determined using BEAMing technology as described inUS Patent Publication No. US20070065823. In another embodiment, therestriction efficiency is determined and the efficiency rate is used tofurther determine the amount of fetal DNA. Exemplary differentiallymethylated nucleic acids are provided in SEQ ID NOs: 1-89.

In another aspect of the technology, a method is provided fordetermining the concentration of fetal DNA in a maternal sample, whereinthe maternal sample comprises differentially methylated maternal andfetal DNA, comprising a) determining the total amount of DNA present inthe maternal sample; b) selectively digesting the maternal DNA in amaternal sample using one or more methylation sensitive restrictionenzymes thereby enriching the fetal DNA; c) determining the amount offetal DNA from step b); and d) comparing the amount of fetal DNA fromstep c) to the total amount of DNA from step a), thereby determining theconcentration of fetal DNA in the maternal sample. The concentration offetal DNA can be used inter alia in conjunction with other fetaldiagnostic methods to improve sensitivity or specificity. In oneembodiment, the method for determining the amount of fetal DNA does notrequire the use of a polymorphic sequence. For example, an allelic ratiois not used to quantify the fetal DNA in step b). In another embodiment,the method for determining the amount of fetal DNA does not require thetreatment of DNA with bisulfite to convert cytosine residues to uracil.In one embodiment, determining the amount of fetal DNA in step b) isdone by introducing one or more competitors at known concentrations. Inanother embodiment, determining the amount of fetal DNA in step b) isdone by RT-PCR, sequencing or counting. In another embodiment, therestriction efficiency is determined and used to further determine theamount of total DNA and fetal DNA. Exemplary differentially methylatednucleic acids are provided in SEQ ID NOs: 1-89.

In another aspect of the technology, a method is provided fordetermining the presence or absence of a fetal aneuploidy using fetalDNA from a maternal sample, wherein the maternal sample comprisesdifferentially methylated maternal and fetal DNA, comprising a)selectively digesting the maternal DNA in a maternal sample using one ormore methylation sensitive restriction enzymes thereby enriching thefetal DNA; b) determining the amount of fetal DNA from a targetchromosome; c) determining the amount of fetal DNA from a referencechromosome; and d) comparing the amount of fetal DNA from step b) tostep c), wherein a biologically or statistically significant differencebetween the amount of target and reference fetal DNA is indicative ofthe presence of a fetal aneuploidy. In one embodiment, the method fordetermining the amount of fetal DNA does not require the use of apolymorphic sequence. For example, an allelic ratio is not used toquantify the fetal DNA in step b). In another embodiment, the method fordetermining the amount of fetal DNA does not require the treatment ofDNA with bisulfite to convert cytosine residues to uracil. In oneembodiment, determining the amount of fetal DNA in steps b) and c) isdone by introducing one or more competitors at known concentrations. Inanother embodiment, determining the amount of fetal DNA in steps b) andc) is done by RT-PCR, sequencing or counting. In another embodiment, theamount of fetal DNA from a target chromosome determined in step b) iscompared to a standard control, for example, the amount of fetal DNAfrom a target chromosome from euploid pregnancies. In anotherembodiment, the restriction efficiency is determined and used to furtherdetermine the amount of fetal DNA from a target chromosome and from areference chromosome. Exemplary differentially methylated nucleic acidsare provided in SEQ ID NOs: 1-89.

In another aspect of the technology, a method is provided for detectingthe presence or absence of a chromosomal abnormality by analyzing theamount or copy number of target nucleic acid and control nucleic acidfrom a sample of differentially methylated nucleic acids comprising thesteps of: (a) enriching a target nucleic acid, from a sample, and acontrol nucleic acid, from the sample, based on its methylation state;(b) performing a copy number analysis of the enriched target nucleicacid in at least one of the fractions; (c) performing a copy numberanalysis of the enriched control nucleic acid in at least one of thefractions; (d) comparing the copy number from step (b) with the copynumber from step (c); and (e) determining if a chromosomal abnormalityexists based on the comparison in step (d), wherein the target nucleicacid and control nucleic acid have the same or substantially the samemethylation status. In a related embodiment, a method is provided fordetecting the presence or absence of a chromosomal abnormality byanalyzing the amount or copy number of target nucleic acid and controlnucleic acid from a sample of differentially methylated nucleic acidscomprising the steps of: (a) binding a target nucleic acid, from asample, and a control nucleic acid, from the sample, to a binding agent;(b) eluting the bound nucleic acid based on methylation status, whereindifferentially methylated nucleic acids elute at least partly intoseparate fractions; (c) performing a copy number analysis of the elutedtarget nucleic acid in at least one of the fractions; (d) performing acopy number analysis of the eluted control nucleic acid in at least oneof the fractions; (e) comparing the copy number from step (c) with thecopy number from step (d); and (f) determining if a chromosomalabnormality exists based on the comparison in step (e), wherein thetarget nucleic acid and control nucleic acid have the same orsubstantially the same methylation status. Differentially methylatednucleic acids are provided in SEQ ID NOs: 1-89.

In another aspect of the technology, a method is provided for detectingthe presence or absence of a chromosomal abnormality by analyzing theallelic ratio of target nucleic acid and control nucleic acid from asample of differentially methylated nucleic acids comprising the stepsof: (a) binding a target nucleic acid, from a sample, and a controlnucleic acid, from the sample, to a binding agent; (b) eluting the boundnucleic acid based on methylation status, wherein differentiallymethylated nucleic acids elute at least partly into separate fractions;(c) performing an allelic ratio analysis of the eluted target nucleicacid in at least one of the fractions; (d) performing an allelic ratioanalysis of the eluted control nucleic acid in at least one of thefractions; (e) comparing the allelic ratio from step c with the allelicratio from step d; and (f) determining if a chromosomal abnormalityexists based on the comparison in step (e), wherein the target nucleicacid and control nucleic acid have the same or substantially the samemethylation status. Differentially methylated nucleic acids are providedin SEQ ID NOs: 1-89, and SNPs within the differentially methylatednucleic acids are provided in Table 2. The methods may also be usefulfor detecting a pregnancy-associated disorder.

In another aspect of the technology, the amount of maternal nucleic acidis determined using the methylation-based methods of the technology. Forexample, fetal nucleic acid can be separated (for example, digestedusing a methylation-sensitive enzyme) from the maternal nucleic acid ina sample, and the maternal nucleic acid can be quantified using themethods of the technology. Once the amount of maternal nucleic acid isdetermined, that amount can subtracted from the total amount of nucleicacid in a sample to determine the amount of fetal nucleic acid. Theamount of fetal nucleic acid can be used to detect fetal traits,including fetal aneuploidy, as described herein.

For aspects and embodiments of the technology described herein, themethods may also be useful for detecting a pregnancy-associateddisorder. In some embodiments, the sample comprises fetal nucleic acid,or fetal nucleic acid and maternal nucleic acid. In the case when thesample comprises fetal and maternal nucleic acid, the fetal nucleic acidand the maternal nucleic acid may have a different methylation status.Nucleic acid species with a different methylation status can bedifferentiated by any method known in the art. In an embodiment, thefetal nucleic acid is enriched by the selective digestion of maternalnucleic acid by a methylation sensitive restriction enzyme. In anotherembodiment, the fetal nucleic acid is enriched by the selectivedigestion of maternal nucleic acid using two or more methylationsensitive restriction enzymes in the same assay. In an embodiment, thetarget nucleic acid and control nucleic acid are both from the fetus. Inanother embodiment, the average size of the fetal nucleic acid is about100 bases to about 500 bases in length. In another embodiment thechromosomal abnormality is an aneuploidy, such as trisomy 21. In someembodiments, the target nucleic acid is at least a portion of achromosome which may be abnormal and the control nucleic acid is atleast a portion of a chromosome which is very rarely abnormal. Forexample, when the target nucleic acid is from chromosome 21, the controlnucleic acid is from a chromosome other than chromosome 21—preferablyanother autosome. In another embodiment, the binding agent is amethylation-specific binding protein such as MBD-Fc. Also, the enrichedor eluted nucleic acid is amplified and/or quantified by any methodknown in the art. In an embodiment, the fetal DNA is quantified using amethod that does not require the use of a polymorphic sequence. Forexample, an allelic ratio is not used to quantify the fetal DNA. Inanother embodiment, the method for quantifying the amount of fetal DNAdoes not require the treatment of DNA with bisulfite to convert cytosineresidues to uracil.

In some embodiments, the methods of the technology include theadditional step of determining the amount of one or moreY-chromosome-specific sequences in a sample. In a related embodiment,the amount of fetal nucleic acid in a sample as determined by using themethylation-based methods of the technology is compared to the amount ofY-chromosome nucleic acid present.

Methods for differentiating nucleic acid based on methylation statusinclude, but are not limited to, methylation sensitive capture, forexample using, MBD2-Fc fragment; bisulfite conversion methods, forexample, MSP (methylation-sensitive PCR), COBRA, methylation-sensitivesingle nucleotide primer extension (Ms-SNuPE) or Sequenom MassCLEAVE™technology; and the use of methylation sensitive restriction enzymes.Except where explicitly stated, any method for differentiating nucleicacid based on methylation status can be used with the compositions andmethods of the technology.

In some embodiments, methods of the technology may further comprise anamplification step. The amplification step can be performed by PCR, suchas methylation-specific PCR. In another embodiment, the amplificationreaction is performed on single molecules, for example, by digital PCR,which is further described in U.S. Pat. Nos. 6,143,496 and 6,440,706,both of which are hereby incorporated by reference. In otherembodiments, the method does not require amplification. For example, theamount of enriched fetal DNA may be determined by counting the fetal DNA(or sequence tags attached thereto) with a flow cytometer or bysequencing means that do not require amplification. In anotherembodiment, the amount of fetal DNA is determined by an amplificationreaction that generates amplicons larger than the digested maternalnucleic acid, thereby further enriching the fetal nucleic acid.

For embodiments that require sequence analysis, any one of the followingsequencing technologies may be used: a primer extension method (e.g.,iPLEX®; Sequenom, Inc.), direct DNA sequencing, restriction fragmentlength polymorphism (RFLP analysis), allele specific oligonucleotide(ASO) analysis, methylation-specific PCR (MSPCR), pyrosequencinganalysis, acycloprime analysis, Reverse dot blot, GeneChip microarrays,Dynamic allele-specific hybridization (DASH), Peptide nucleic acid (PNA)and locked nucleic acids (LNA) probes, TaqMan, Molecular Beacons,Intercalating dye, FRET primers, fluorescence tagged dNTP/ddNTPs,AlphaScreen, SNPstream, genetic bit analysis (GBA), Multiplexminisequencing, SNaPshot, GOOD assay, Microarray miniseq, arrayed primerextension (APEX), Microarray primer extension, Tag arrays, Codedmicrospheres, Template-directed incorporation (TDI), fluorescencepolarization, Colorimetric oligonucleotide ligation assay (OLA),Sequence-coded OLA, Microarray ligation, Ligase chain reaction, Padlockprobes, Invader™ assay, hybridization using at least one probe,hybridization using at least one fluorescently labeled probe,electrophoresis, cloning and sequencing, for example as performed on the454 platform (Roche) (Margulies, M. et al. 2005 Nature 437, 376-380),IIlumina Genome Analyzer (or Solexa platform) or SOLiD System (AppliedBiosystems) or the Helicos True Single Molecule DNA sequencingtechnology (Harris T D et al. 2008 Science, 320, 106-109), the singlemolecule, real-time (SMRT™) technology of Pacific Biosciences, ornanopore-based sequencing (Soni GV and Meller A. 2007 Clin Chem 53:1996-2001) and combinations thereof. Nanopore-based methods may includesequencing nucleic acid using a nanopore, or counting nucleic acidmolecules using a nanopore, for example, based on size wherein sequenceinformation is not determined.

The absolute copy number of one or more nucleic acids can be determined,for example, using mass spectrometry, a system that uses a competitivePCR approach for absolute copy number measurements. See for example,Ding C, Cantor C R (2003) A high-throughput gene expression analysistechnique using competitive PCR and matrix-assisted laser desorptionionization time-of-flight MS. Proc Natl Acad Sci USA 100:3059-3064, andU.S. patent application Ser. No. 10/655,762, which published as USPatent Publication No. 20040081993, both of which are herebyincorporated by reference.

In some embodiments, the amount of the genomic sequence is compared witha standard control, wherein an increase or decrease from the standardcontrol indicates the presence or progression of a pregnancy-associateddisorder. For example, the amount of fetal nucleic acid may be comparedto the total amount of DNA present in the sample. Or when detecting thepresence or absence of fetal aneuploidy, the amount of fetal nucleicacid from target chromosome may be compared to the amount of fetalnucleic acid from a reference chromosome. Preferably the referencechromosome is another autosome that has a low rate of aneuploidy. Theratio of target fetal nucleic acid to reference fetal nucleic acid maybe compared to the same ratio from a normal, euploid pregnancy. Forexample, a control ratio may be determined from a DNA sample obtainedfrom a female carrying a healthy fetus who does not have a chromosomalabnormality. Preferably, one uses a panel of control samples. Wherecertain chromosome anomalies are known, one can also have standards thatare indicative of a specific disease or condition. Thus, for example, toscreen for three different chromosomal aneuploidies in a maternal plasmaof a pregnant female, one preferably uses a panel of control DNAs thathave been isolated from mothers who are known to carry a fetus with, forexample, chromosome 13, 18, or 21 trisomy, and a mother who is pregnantwith a fetus who does not have a chromosomal abnormality.

In some embodiments, the present technology provides a method in whichthe alleles from the target nucleic acid and control nucleic acid aredifferentiated by sequence variation. The sequence variation may be asingle nucleotide polymorphism (SNP) or an insertion/deletionpolymorphism. In an embodiment, the fetal nucleic acid should compriseat least one high frequency heterozygous polymorphism (e.g., about 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 25% or more frequency rate), which allows the determination ofthe allelic-ratio of the nucleic acid in order to assess the presence orabsence of the chromosomal abnormality. A list of exemplary SNPs isprovided in Table 2, however, this does not represent a complete list ofpolymorphic alleles that can be used as part of the technology. Any SNPmeeting the following criteria may also be considered: (a) the SNP has aheterozygosity frequency greater than about 2% (preferably across arange of different populations), (b) the SNP is a heterozygous locus;and (c)(i) the SNP is within nucleic acid sequence described herein, or(c)(iii) the SNP is within about 5 to about 2000 base pairs of a SNPdescribed herein (e.g., within about 5, 10, 15, 20, 25, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250,1500, 1750 or 2000 base pairs of a SNP described herein).

In other embodiments, the sequence variation is a short tandem repeat(STR) polymorphism. In some embodiments, the sequence variation falls ina restriction site, whereby one allele is susceptible to digestion by arestriction enzyme and the one or more other alleles are not. In someembodiments, the sequence variation is a methylation site.

In some embodiments, performing an allelic ratio analysis comprisesdetermining the ratio of alleles of the target nucleic acid and controlnucleic acid from the fetus of a pregnant woman by obtaining an nucleicacid-containing biological sample from the pregnant woman, wherein thebiological sample contains fetal nucleic acid, partially or whollyseparating the fetal nucleic acid from the maternal nucleic acid basedon differential methylation, discriminating the alleles from the targetnucleic acid and the control nucleic acid, followed by determination ofthe ratio of the alleles, and detecting the presence or absence of achromosomal disorder in the fetus based on the ratio of alleles, whereina ratio above or below a normal, euploid ratio is indicative of achromosomal disorder. In one embodiment, the target nucleic acid is froma suspected aneuploid chromosome (e.g., chromosome 21) and the controlnucleic acid is from a euploid chromosome from the same fetus.

In some embodiments, the present technology is combined with other fetalmarkers to detect the presence or absence of multiple chromosomalabnormalities, wherein the chromosomal abnormalities are selected fromthe following: trisomy 21, trisomy 18 and trisomy 13, or combinationsthereof. In some embodiments, the chromosomal disorder involves the Xchromosome or the Y chromosome.

In some embodiments, the compositions or processes may be multiplexed ina single reaction. For example, the amount of fetal nucleic acid may bedetermined at multiple loci across the genome. Or when detecting thepresence or absence of fetal aneuploidy, the amount of fetal nucleicacid may be determined at multiple loci on one or more targetchromosomes (e.g., chromosomes 13, 18 or 21) and on one or morereference chromosomes. If an allelic ratio is being used, one or morealleles from Table 2 can be detected and discriminated simultaneously.When determining allelic ratios, multiplexing embodiments areparticularly important when the genotype at a polymorphic locus is notknown. In some instances, for example when the mother and child arehomozygous at the polymorphic locus, the assay may not be informative.In one embodiment, greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 100, 200, 300 or500, and any intermediate levels, polynucleotide sequences of thetechnology are enriched, separated and/or examined according the methodsof the technology. When detecting a chromosomal abnormality by analyzingthe copy number of target nucleic acid and control nucleic acid, lessthan 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 polynucleotide sequencesmay need to be analyzed to accurately detect the presence or absence ofa chromosomal abnormality. In another embodiment, the compositions orprocesses of the technology may be used to assay samples that have beendivided into 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 50, 100 or more replicates, or into singlemolecule equivalents. Methods for analyzing fetal nucleic acids from amaternal sample in replicates, including single molecule analyses, areprovided in US application Ser. No. 11/364,294, which published as USPatent Publication No. US 2007-0207466 A1, which is hereby incorporatedby reference.

In a further embodiment, the present technology provides a methodwherein a comparison step shows an increased risk of a fetus having achromosomal disorder if the ratio of the alleles or absolute copy numberof the target nucleic acid is higher or lower by 1 standard deviationfrom the standard control sequence. In some embodiments, the comparisonstep shows an increased risk of a fetus having a chromosomal disorder ifthe ratio of the alleles or absolute copy number of the target nucleicacid is higher or lower by 2 standard deviation from the standardcontrol sequence. In some other embodiments, the comparison step showsan increased risk of a fetus having a chromosomal disorder if the ratioof the alleles or absolute copy number of the target nucleic acid ishigher or lower by 3 standard deviation from the standard controlsequence. In some embodiments, the comparison step shows an increasedrisk of a fetus having a chromosomal disorder if the ratio of thealleles or absolute copy number of the target nucleic acid is higher orlower than a statistically significant standard deviation from thecontrol. In one embodiment, the standard control is a maternalreference, and in another embodiment the standard control is a fetalreference chromosome (e.g., non-trisomic autosome).

In some embodiments, the methods of the technology may be combined withother methods for diagnosing a chromosomal abnormality. For example, anoninvasive diagnostic method may require confirmation of the presenceor absence of fetal nucleic acid, such as a sex test for a female fetusor to confirm an RhD negative female fetus in an RhD negative mother. Inanother embodiment, the compositions and methods of the technology maybe used to determine the percentage of fetal nucleic acid in a maternalsample in order to enable another diagnostic method that requires thepercentage of fetal nucleic acid be known. For example, does a samplemeet certain threshold concentration requirements? When determining anallelic ratio to diagnose a fetal aneuploidy from a maternal sample, theamount or concentration of fetal nucleic acid may be required to make adiagnose with a given sensitivity and specificity. In other embodiments,the compositions and methods of the technology for detecting achromosomal abnormality can be combined with other known methods therebyimproving the overall sensitivity and specificity of the detectionmethod. For example, mathematical models have suggested that a combinedfirst-trimester screening program utilizing maternal age (MA), nuchaltranslucency (NT) thickness, serum-free beta-hCG, and serum PAPP-A willdetect more than 80% of fetuses with Down's syndrome for a 5% invasivetesting rate (Wald and Hackshaw, Prenat Diagn 17(9):921-9 (1997)).However, the combination of commonly used aneuploidy detection methodscombined with the non-invasive free fetal nucleic acid-based methodsdescribed herein may offer improved accuracy with a lower false positiverate. Examples of combined diagnostic methods are provided in PCTPublication Number WO2008157264A2 (assigned to the Applicant), which ishereby incorporated by reference. In some embodiments, the methods ofthe technology may be combined with cell-based methods, wherein fetalcells are procured invasively or non-invasively.

In certain embodiments, an increased risk for a chromosomal abnormalityis based on the outcome or result(s) produced from the compositions ormethods provided herein. An example of an outcome is a deviation fromthe euploid absolute copy number or allelic ratio, which indicates thepresence of chromosomal aneuploidy. This increase or decrease in theabsolute copy number or ratio from the standard control indicates anincreased risk of having a fetus with a chromosomal abnormality (e.g.,trisomy 21). Information pertaining to a method described herein, suchas an outcome, result, or risk of trisomy or aneuploidy, for example,may be transfixed, renditioned, recorded and/or displayed in anysuitable medium. For example, an outcome may be transfixed in a mediumto save, store, share, communicate or otherwise analyze the outcome. Amedium can be tangible (e.g., paper) or intangible (e.g., electronicmedium), and examples of media include, but are not limited to, computermedia, databases, charts, patient charts, records, patient records,graphs and tables, and any other medium of expression. The informationsometimes is stored and/or renditioned in computer readable form andsometimes is stored and organized in a database. In certain embodiments,the information may be transferred from one location to another using aphysical medium (e.g., paper) or a computer readable medium (e.g.,optical and/or magnetic storage or transmission medium, floppy disk,hard disk, random access memory, computer processing unit, facsimilesignal, satellite signal, transmission over an internet or transmissionover the world-wide web).

A CpG island may be used as the CpG-containing genomic sequence in somecases, whereas in other cases the CpG-containing genomic sequence maynot be a CpG island. In some embodiments, the present technologyprovides a kit for performing the methods of the technology. Onecomponent of the kit is a methylation-sensitive binding agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows the design of the recombinant MBD-Fc protein used toseparate differentially methylated DNA.

FIG. 2: Shows the methyl-CpG-binding, antibody-like protein has a highaffinity and high avidity to its “antigen”, which is preferably DNA thatis methylated at CpG di-nucleotides.

FIG. 3: Shows the methyl binding domain of MBD-FC binds DNA moleculesregardless of their methylation status. The strength of this protein/DNAinteraction is defined by the level of DNA methylation. After bindinggenomic DNA, eluate solutions of increasing salt concentrations can beused to fractionate non-methylated and methylated DNA allowing for acontrolled separation.

FIG. 4: Shows the experiment used to identify differentially methylatedDNA from a fetus and mother using the recombinant MBD-Fc protein and amicroarray.

FIG. 5: Shows typical results generated by Sequenom® EpiTYPER™ method,which was used to validate the results generated from the experimentillustrated in FIG. 4.

FIG. 6: Shows the correlation between the log ratios derived frommicroarray analysis (x axis) and methylation differences obtained byEpiTYPER analysis (y axis). Each data point represents the average forone region across all measured samples. The microarray analysis iscomparative in nature because the highly methylated fraction of thematernal DNA is hybridized together with the highly methylated fractionof placenta DNA. Positive values indicate higher methylation of theplacenta samples. In mass spectrometry each samples is measuredindividually. We first calculated difference in methylation bysubtracting the maternal methylation values from the placentamethylation value. To compare the results with the microarray data wecalculated the average of the differences for all maternal/placenta DNApairs. FIG. 6 discloses SEQ ID NOS 178-179, respectively, in order ofappearance.

FIG. 8: Shown is the correlation between the number of gDNA moleculesthat were expected and the number of molecules measured by competitivePCR in combination with mass spectrometry analysis. In this experimentwe used DNA derived from whole blood (black plus signs) and commerciallyavailable fully methylated DNA (red crosses) in a 90 to 10 ratio. Weused the MBD-FC fusion protein to separate the non-methylated and themethylated fraction of DNA. Each fraction was subject to competitive PCRanalysis with mass spectrometry readout. The method has been describedearlier for the analysis of copy number variations and is commerciallyavailable for gene expression analysis. The approach allows absolutequantification of DNA molecules with the help of a syntheticoligonucleotides of know concentration. In this experiment we targetedthe MGMT locus, which was not methylated in the whole blood sample usedhere. Using an input of 300 total gDNA copies we expect to see 270copies of non-methylated DNA and 30 copies of methylated DNA. Themeasured copy numbers are largely in agreement with the expected values.The data point at 600 copies of input DNA indicates a bias in thereaction and shows that this initial proof of concept experiment needsto be followed up with more development work, before the assay can beused. However, this initial data indicates the feasibility of theapproach for capturing and quantifying of a few copies of methylated DNAin the presence of an excess of unmethylated DNA species.

FIG. 9A-9C: Shown are bar graph plots of the methylation differencesobtained from the microarray analysis (dark bars) and the massspectrometry analysis (light grey bars) with respect to their genomiclocation. For each of the 85 region that were identified to bedifferentially methylated by microarray an individual plot is provided.The x axis for each plot shows the chromosomal position of the region.The y axis depicts the log ration (in case of the microarrays) and themethylation differences (in case of the mass spectrometry results). Forthe microarrays each hybridization probe in the area is shown as asingle black (or dark grey) bar. For the mass spectrometry results eachCpG site, is shown as a light grey bar. Bars showing values greater thanzero indicate higher DNA methylation in the placenta samples compared tothe maternal DNA. For some genes the differences are small (i.e. RBI orDSCR6) but still statistically significant. Those regions would be lesssuitable for a fetal DNA enrichment strategy.

FIG. 10: Shows one embodiment of the Fetal Quantifier Method. Maternalnucleic acid is selectively digested and the remaining fetal nucleicacid is quantified using a competitor of known concentration. In thisschema, the analyte is separated and quantified by a mass spectrometer.

FIG. 11: Shows one embodiment of the Methylation-Based Fetal DiagnosticMethod. Maternal nucleic acid is selectively digested and the remainingfetal nucleic acid is quantified for three different chromosomes (13, 18and 21). Parts 2 and 3 of the Figure illustrate the size distribution ofthe nucleic acid in the sample before and after digestion. Theamplification reactions can be size-specific (e.g., greater than 100base pair amplicons) such that they favor the longer, non-digested fetalnucleic acid over the digested maternal nucleic acid, thereby furtherenriching the fetal nucleic acid. The spectra at the bottom of theFigure show an increased amount of chromosome 21 fetal nucleic acidindicative of trisomy 21.

FIG. 12: Shows the total number of amplifiable genomic copies from fourdifferent DNA samples isolated from the blood of non-pregnant women.Each sample was diluted to contain approximately 2500, 1250, 625 or 313copies per reaction. Each measurement was obtained by taking the meanDNA/competitor ratio obtained from two total copy number assays (ALB andRNAseP in Table X). As FIG. 12 shows, the total copy number is accurateand stable across the different samples, thus validating the usefulnessof the competitor-based approach.

FIGS. 13A and B: A model system was created that contained a constantnumber of maternal non-methylated DNA with varying amounts of maleplacental methylated DNA spiked-in. The samples were spiked with maleplacental amounts ranging from approximately 0 to 25% relative to thematernal non-methylated DNA. The fraction of placental DNA wascalculated using the ratios obtained from the methylation assays (FIG.13A) and the Y-chromosome marker (FIG. 13B) as compared to the totalcopy number assay. The methylation and Y-chromosome markers are providedin Table X.

FIGS. 14 A and B: Show the results of the total copy number assay fromplasma samples. In FIG. 14A, the copy number for each sample is shown.Two samples (no 25 and 26) have a significantly higher total copy numberthan all the other samples. A mean of approximately 1300 amplifiablecopies/ml plasma was obtained (range 766-2055). FIG. 14B shows abox-and-whisker plot of the given values, summarizing the results.

FIGS. 15A and B: The amount (or copy numbers) of fetal nucleic acid from33 different plasma samples taken from pregnant women with male fetusesare plotted. The copy numbers obtained were calculated using themethylation markers and the Y-chromosome-specific markers using theassays provided in Table X. As can be seen in FIG. 15B, thebox-and-whisker plot of the given values indicated minimal differencebetween the two different measurements, thus validating the accuracy andstability of the method.

FIG. 16: Shows a paired correlation between the results obtained usingthe methylation markers versus the Y-chromosome marker from FIG. 15A.

FIG. 17: Shows the digestion efficiency of the restriction enzymes usingthe ratio of digestion for the control versus the competitor andcomparing this value to the mean total copy number assays. Apart fromsample 26 all reactions indicate the efficiency to be above about 99%.

FIG. 18: Provides a specific method for calculating fetal DNA fraction(or concentration) in a sample using the Y-chromosome-specific markersfor male pregnancies and the mean of the methylated fraction for allpregnancies (regardless of fetal sex).

FIG. 19: Provides a specific method for calculating fetal DNA fraction(or concentration) in a sample without the Y-chromosome-specificmarkers. Instead, only the Assays for Methylation Quantification wereused to determine the concentration of fetal DNA.

FIG. 20: Shows a power calculation t-test for a simulated trisomy 21diagnosis using the methods of the technology. The Figure shows therelationship between the coefficient of variation (CV) on the x-axis andthe power to discriminate the assay populations using a simple t-test(y-axis). The data indicates that in 99% of all cases, one candiscriminate the two population (euploid vs. aneuploid) on asignificance level of 0.001 provided a CV of 5% or less.

DEFINITIONS

The term “pregnancy-associated disorder,” as used in this application,refers to any condition or disease that may affect a pregnant woman, thefetus, or both the woman and the fetus. Such a condition or disease maymanifest its symptoms during a limited time period, e.g., duringpregnancy or delivery, or may last the entire life span of the fetusfollowing its birth. Some examples of a pregnancy-associated disorderinclude ectopic pregnancy, preeclampsia, preterm labor, RhDincompatibility, fetal chromosomal abnormalities such as trisomy 21, andgenetically inherited fetal disorders such as cystic fibrosis,beta-thalassemia or other monogenic disorders. The ability to enrichfetal nucleic from a maternal sample may prove particularly useful forthe noninvasive prenatal diagnosis of autosomal recessive diseases suchas the case when a mother and father share an identical disease causingmutation, an occurrence previously perceived as a challenge for maternalplasma-based non-trisomy prenatal diagnosis.

The term “chromosomal abnormality” or “aneuploidy” as used herein refersto a deviation between the structure of the subject chromosome and anormal homologous chromosome. The term “normal” refers to thepredominate karyotype or banding pattern found in healthy individuals ofa particular species, for example, a euploid genome (in humans, 46XX or46XY). A chromosomal abnormality can be numerical or structural, andincludes but is not limited to aneuploidy, polyploidy, inversion, atrisomy, a monosomy, duplication, deletion, deletion of a part of achromosome, addition, addition of a part of chromosome, insertion, afragment of a chromosome, a region of a chromosome, chromosomalrearrangement, and translocation. Chromosomal abnormality may also referto a state of chromosomal abnormality where a portion of one or morechromosomes is not an exact multiple of the usual haploid number due to,for example, chromosome translocation. Chromosomal translocation (e.g.translocation between chromosome 21 and 14 where some of the 14thchromosome is replaced by extra 21st chromosome) may cause partialtrisomy 21. A chromosomal abnormality can be correlated with presence ofa pathological condition or with a predisposition to develop apathological condition. A chromosomal abnormality may be detected byquantitative analysis of nucleic acid.

The terms “nucleic acid” and “nucleic acid molecule” may be usedinterchangeably throughout the disclosure. The terms refer to nucleicacids of any composition from, such as DNA (e.g., complementary DNA(cDNA), genomic DNA (gDNA) and the like), RNA (e.g., message RNA (mRNA),short inhibitory RNA (siRNA), ribosomal RNA (rRNA), tRNA, microRNA, RNAhighly expressed by the fetus or placenta, and the like), and/or DNA orRNA analogs (e.g., containing base analogs, sugar analogs and/or anon-native backbone and the like), RNA/DNA hybrids and polyamide nucleicacids (PNAs), all of which can be in single- or double-stranded form,and unless otherwise limited, can encompass known analogs of naturalnucleotides that can function in a similar manner as naturally occurringnucleotides. For example, the nucleic acids provided in SEQ ID Nos: 1-89(see Table 4) can be in any form useful for conducting processes herein(e.g., linear, circular, supercoiled, single-stranded, double-strandedand the like) or may include variations (e.g., insertions, deletions orsubstitutions) that do not alter their utility as part of the presenttechnology. A nucleic acid may be, or may be from, a plasmid, phage,autonomously replicating sequence (ARS), centromere, artificialchromosome, chromosome, or other nucleic acid able to replicate or bereplicated in vitro or in a host cell, a cell, a cell nucleus orcytoplasm of a cell in certain embodiments. A template nucleic acid insome embodiments can be from a single chromosome (e.g., a nucleic acidsample may be from one chromosome of a sample obtained from a diploidorganism). Unless specifically limited, the term encompasses nucleicacids containing known analogs of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions), alleles, orthologs, single nucleotidepolymorphisms (SNPs), and complementary sequences as well as thesequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Theterm nucleic acid is used interchangeably with locus, gene, cDNA, andmRNA encoded by a gene. The term also may include, as equivalents,derivatives, variants and analogs of RNA or DNA synthesized fromnucleotide analogs, single-stranded (“sense” or “antisense”, “plus”strand or “minus” strand, “forward” reading frame or “reverse” readingframe) and double-stranded polynucleotides. Deoxyribonucleotides includedeoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. ForRNA, the base cytosine is replaced with uracil. A template nucleic acidmay be prepared using a nucleic acid obtained from a subject as atemplate.

A “nucleic acid comprising one or more CpG sites” or a “CpG-containinggenomic sequence” as used herein refers to a segment of DNA sequence ata defined location in the genome of an individual such as a human fetusor a pregnant woman. Typically, a “CpG-containing genomic sequence” isat least 15 nucleotides in length and contains at least one cytosine.Preferably, it can be at least 30, 50, 80, 100, 150, 200, 250, or 300nucleotides in length and contains at least 2, 5, 10, 15, 20, 25, or 30cytosines. For anyone “CpG-containing genomic sequence” at a givenlocation, e.g., within a region centering around a given genetic locus(see Table 1), nucleotide sequence variations may exist from individualto individual and from allele to allele even for the same individual.Typically, such a region centering around a defined genetic locus (e.g.,a CpG island) contains the locus as well as upstream and/or downstreamsequences. Each of the upstream or downstream sequence (counting fromthe 5′ or 3′ boundary of the genetic locus, respectively) can be as longas 10 kb, in other cases may be as long as 5 kb, 2 kb, 1 kb, 500 bp, 200bp, or 100 bp. Furthermore, a “CpG-containing genomic sequence” mayencompass a nucleotide sequence transcribed or not transcribed forprotein production, and the nucleotide sequence can be an inter-genesequence, intra-gene sequence, protein-coding sequence, a nonprotein-coding sequence (such as a transcription promoter), or acombination thereof.

As used herein, a “methylated nucleotide” or a “methylated nucleotidebase” refers to the presence of a methyl moiety on a nucleotide base,where the methyl moiety is not present in a recognized typicalnucleotide base. For example, cytosine does not contain a methyl moietyon its pyrimidine ring, but 5-methylcytosine contains a methyl moiety atposition 5 of its pyrimidine ring. Therefore, cytosine is not amethylated nucleotide and 5-methylcytosine is a methylated nucleotide.In another example, thymine contains a methyl moiety at position 5 ofits pyrimidine ring, however, for purposes herein, thymine is notconsidered a methylated nucleotide when present in DNA since thymine isa typical nucleotide base of DNA. Typical nucleoside bases for DNA arethymine, adenine, cytosine and guanine. Typical bases for RNA areuracil, adenine, cytosine and guanine. Correspondingly a “methylationsite” is the location in the target gene nucleic acid region wheremethylation has, or has the possibility of occurring. For example alocation containing CpG is a methylation site wherein the cytosine mayor may not be methylated.

As used herein, a “CpG site” or “methylation site” is a nucleotidewithin a nucleic acid that is susceptible to methylation either bynatural occurring events in vivo or by an event instituted to chemicallymethylate the nucleotide in vitro.

As used herein, a “methylated nucleic acid molecule” refers to a nucleicacid molecule that contains one or more methylated nucleotides thatis/are methylated.

A “CpG island” as used herein describes a segment of DNA sequence thatcomprises a functionally or structurally deviated CpG density. Forexample, Yamada et al. (Genome Research 14:247-266, 2004) have describeda set of standards for determining a CpG island: it must be at least 400nucleotides in length, has a greater than 50% GC content, and an OCF/ECFratio greater than 0.6. Others (Takai et al., Proc. Natl. Acad. Sci.U.S.A. 99:3740-3745, 2002) have defined a CpG island less stringently asa sequence at least 200 nucleotides in length, having a greater than 50%GC content, and an OCF/ECF ratio greater than 0.6.

The term “epigenetic state” or “epigenetic status” as used herein refersto any structural feature at a molecular level of a nucleic acid (e.g.,DNA or RNA) other than the primary nucleotide sequence. For instance,the epigenetic state of a genomic DNA may include its secondary ortertiary structure determined or influenced by, e.g., its methylationpattern or its association with cellular proteins.

The term “methylation profile” “methylation state” or “methylationstatus,” as used herein to describe the state of methylation of agenomic sequence, refers to the characteristics of a DNA segment at aparticular genomic locus relevant to methylation. Such characteristicsinclude, but are not limited to, whether any of the cytosine (C)residues within this DNA sequence are methylated, location of methylatedC residue(s), percentage of methylated C at any particular stretch ofresidues, and allelic differences in methylation due to, e.g.,difference in the origin of the alleles. The term “methylation” profile”or “methylation status” also refers to the relative or absoluteconcentration of methylated C or unmethylated C at any particularstretch of residues in a biological sample. For example, if the cytosine(C) residue(s) within a DNA sequence are methylated it may be referredto as “hypermethylated”; whereas if the cytosine (C) residue(s) within aDNA sequence are not methylated it may be referred to as“hypomethylated”. Likewise, if the cytosine (C) residue(s) within a DNAsequence (e.g., fetal nucleic acid) are methylated as compared toanother sequence from a different region or from a different individual(e.g., relative to maternal nucleic acid), that sequence is consideredhypermethylated compared to the other sequence. Alternatively, if thecytosine (C) residue(s) within a DNA sequence are not methylated ascompared to another sequence from a different region or from a differentindividual (e.g., the mother), that sequence is consideredhypomethylated compared to the other sequence. These sequences are saidto be “differentially methylated”, and more specifically, when themethylation status differs between mother and fetus, the sequences areconsidered “differentially methylated maternal and fetal nucleic acid”.

The term “agent that binds to methylated nucleotides” as used hereinrefers to a substance that is capable of binding to methylated nucleicacid. The agent may be naturally-occurring or synthetic, and may bemodified or unmodified. In one embodiment, the agent allows for theseparation of different nucleic acid species according to theirrespective methylation states. An example of an agent that binds tomethylated nucleotides is described in PCT Patent Application No.PCT/EP2005/012707, which published as WO06056480A2 and is herebyincorporated by reference. The described agent is a bifunctionalpolypeptide comprising the DNA-binding domain of a protein belonging tothe family of Methyl-CpG binding proteins (MBDs) and an Fc portion of anantibody (see FIG. 1). The recombinant methyl-CpG-binding, antibody-likeprotein can preferably bind CpG methylated DNA in an antibody-likemanner. That means, the methyl-CpG-binding, antibody-like protein has ahigh affinity and high avidity to its “antigen”, which is preferably DNAthat is methylated at CpG dinucleotides. The agent may also be amultivalent MBD (see FIG. 2).

The term “polymorphism” as used herein refers to a sequence variationwithin different alleles of the same genomic sequence. A sequence thatcontains a polymorphism is considered “polymorphic sequence”. Detectionof one or more polymorphisms allows differentiation of different allelesof a single genomic sequence or between two or more individuals. As usedherein, the term “polymorphic marker” or “polymorphic sequence” refersto segments of genomic DNA that exhibit heritable variation in a DNAsequence between individuals. Such markers include, but are not limitedto, single nucleotide polymorphisms (SNPs), restriction fragment lengthpolymorphisms (RFLPs), short tandem repeats, such as di-, tri- ortetra-nucleotide repeats (STRs), and the like. Polymorphic markersaccording to the present technology can be used to specificallydifferentiate between a maternal and paternal allele in the enrichedfetal nucleic acid sample.

The terms “single nucleotide polymorphism” or “SNP” as used herein referto the polynucleotide sequence variation present at a single nucleotideresidue within different alleles of the same genomic sequence. Thisvariation may occur within the coding region or non-coding region (i.e.,in the promoter or intronic region) of a genomic sequence, if thegenomic sequence is transcribed during protein production. Detection ofone or more SNP allows differentiation of different alleles of a singlegenomic sequence or between two or more individuals.

The term “allele” as used herein is one of several alternate forms of agene or non-coding regions of DNA that occupy the same position on achromosome. The term allele can be used to describe DNA from anyorganism including but not limited to bacteria, viruses, fungi,protozoa, molds, yeasts, plants, humans, non-humans, animals, andarcheabacteria.

The terms “ratio of the alleles” or “allelic ratio” as used herein referto the ratio of the population of one allele and the population of theother allele in a sample. In some trisomic cases, it is possible that afetus may be tri-allelic for a particular locus. In such cases, the term“ratio of the alleles” refers to the ratio of the population of any oneallele against one of the other alleles, or any one allele against theother two alleles.

The term “non-polymorphism-based quantitative method” as used hereinrefers to a method for determining the amount of an analyte (e.g., totalnucleic acid, Y-chromosome nucleic acid, or fetal nucleic acid) thatdoes not require the use of a polymorphic marker or sequence. Although apolymorphism may be present in the sequence, said polymorphism is notrequired to quantify the sequence. Examples of non-polymorphism-basedquantitative methods include, but are not limited to, RT-PCR, digitalPCR, array-based methods, sequencing methods, nanopore-based methods,nucleic acid-bound bead-based counting methods and competitor-basedmethods wherein one or more competitors are introduced at a knownconcentration(s) to determine the amount of one or more analytes. Insome embodiments, some of the above exemplary methods (for example,sequencing) may need to be actively modified or designed such that oneor more polymorphisms are not interrogated.

The terms “absolute amount” or “copy number” as used herein refers tothe amount or quantity of an analyte (e.g., total nucleic acid or fetalnucleic acid). The present technology provides compositions andprocesses for determining the absolute amount of fetal nucleic acid in amixed maternal sample. Absolute amount or copy number represents thenumber of molecules available for detection, and may be expressed as thegenomic equivalents per unit. The term “concentration” refers to theamount or proportion of a substance in a mixture or solution (e.g., theamount of fetal nucleic acid in a maternal sample that comprises amixture of maternal and fetal nucleic acid). The concentration may beexpressed as a percentage, which is used to express how large/small onequantity is, relative to another quantity as a fraction of 100.Platforms for determining the quantity or amount of an analyte (e.g.,target nucleic acid) include, but are not limited to, massspectrometery, digital PCR, sequencing by synthesis platforms (e.g.,pyrosequencing), fluorescence spectroscopy and flow cytometry.

The term “sample” as used herein refers to a specimen containing nucleicacid. Examples of samples include, but are not limited to, tissue,bodily fluid (for example, blood, serum, plasma, saliva, urine, tears,peritoneal fluid, ascitic fluid, vaginal secretion, breast fluid, breastmilk, lymph fluid, cerebrospinal fluid or mucosa secretion), umbilicalcord blood, chorionic villi, amniotic fluid, an embryo, a two-celledembryo, a four-celled embryo, an eight-celled embryo, a 16-celledembryo, a 32-celled embryo, a 64-celled embryo, a 128-celled embryo, a256-celled embryo, a 512-celled embryo, a 1024-celled embryo, embryonictissues, lymph fluid, cerebrospinal fluid, mucosa secretion, or otherbody exudate, fecal matter, an individual cell or extract of the suchsources that contain the nucleic acid of the same, and subcellularstructures such as mitochondria, using protocols well established withinthe art.

Fetal DNA can be obtained from sources including but not limited tomaternal blood, maternal serum, maternal plasma, fetal cells, umbilicalcord blood, chorionic villi, amniotic fluid, urine, saliva, lung lavage,cells or tissues.

The term “blood” as used herein refers to a blood sample or preparationfrom a pregnant woman or a woman being tested for possible pregnancy.The term encompasses whole blood or any fractions of blood, such asserum and plasma as conventionally defined.

The term “bisulfite” as used herein encompasses any suitable types ofbisulfites, such as sodium bisulfite, that are capable of chemicallyconverting a cytosine (C) to a uracil (U) without chemically modifying amethylated cytosine and therefore can be used to differentially modify aDNA sequence based on the methylation status of the DNA.

As used herein, a reagent that “differentially modifies” methylated ornon-methylated DNA encompasses any reagent that modifies methylatedand/or unmethylated DNA in a process through which distinguishableproducts result from methylated and non-methylated DNA, thereby allowingthe identification of the DNA methylation status. Such processes mayinclude, but are not limited to, chemical reactions (such as aC.fwdarw.0 conversion by bisulfite) and enzymatic treatment (such ascleavage by a methylation-dependent endonuclease). Thus, an enzyme thatpreferentially cleaves or digests methylated DNA is one capable ofcleaving or digesting a DNA molecule at a much higher efficiency whenthe DNA is methylated, whereas an enzyme that preferentially cleaves ordigests unmethylated DNA exhibits a significantly higher efficiency whenthe DNA is not methylated.

The terms “non-bisulfite-based method” and “non-bisulfite-basedquantitative method” as used herein refer to any method for quantifyingmethylated or non-methylated nucleic acid that does not require the useof bisulfite. The terms also refer to methods for preparing a nucleicacid to be quantified that do not require bisulfite treatment. Examplesof non-bisulfite-based methods include, but are not limited to, methodsfor digesting nucleic acid using one or more methylation sensitiveenzymes and methods for separating nucleic acid using agents that bindnucleic acid based on methylation status.

The terms “methyl-sensitive enzymes” and “methylation sensitiverestriction enzymes” are DNA restriction endonucleases that aredependent on the methylation state of their DNA recognition site foractivity. For example, there are methyl-sensitive enzymes that cleave ordigest at their DNA recognition sequence only if it is not methylated.Thus, an unmethylated DNA sample will be cut into smaller fragments thana methylated DNA sample. Similarly, a hypermethylated DNA sample willnot be cleaved. In contrast, there are methyl-sensitive enzymes thatcleave at their DNA recognition sequence only if it is methylated. Asused herein, the terms “cleave”, “cut” and “digest” are usedinterchangeably.

The term “target nucleic acid” as used herein refers to a nucleic acidexamined using the methods disclosed herein to determine if the nucleicacid is part of a pregnancy-related disorder or chromosomal abnormality.For example, a target nucleic acid from chromosome 21 could be examinedusing the methods of the technology to detect Down's Syndrome.

The term “control nucleic acid” as used herein refers to a nucleic acidused as a reference nucleic acid according to the methods disclosedherein to determine if the nucleic acid is part of a chromosomalabnormality. For example, a control nucleic acid from a chromosome otherthan chromosome 21 (herein referred to as a “reference chromosome”)could be as a reference sequence to detect Down's Syndrome. In someembodiments, the control sequence has a known or predetermined quantity.

The term “sequence-specific” or “locus-specific method” as used hereinrefers to a method that interrogates (for example, quantifies) nucleicacid at a specific location (or locus) in the genome based on thesequence composition. Sequence-specific or locus-specific methods allowfor the quantification of specific regions or chromosomes.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) involved in thetranscription/translation of the gene product and the regulation of thetranscription/translation, as well as intervening sequences (introns)between individual coding segments (exons).

In this application, the terms “polypeptide,” “peptide,” and “protein”are used interchangeably herein to refer to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull-length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, .gamma.-carboxyglutamate, and O-phosphoserine.

Amino acids may be referred to herein by either the commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

“Primers” as used herein refer to oligonucleotides that can be used inan amplification method, such as a polymerase chain reaction (PCR), toamplify a nucleotide sequence based on the polynucleotide sequencecorresponding to a particular genomic sequence, e.g., one located withinthe CpG island CGI137, PDE9A, or CGI009 on chromosome 21, in variousmethylation status. At least one of the PCR primers for amplification ofa polynucleotide sequence is sequence-specific for the sequence.

The term “template” refers to any nucleic acid molecule that can be usedfor amplification in the technology. RNA or DNA that is not naturallydouble stranded can be made into double stranded DNA so as to be used astemplate DNA. Any double stranded DNA or preparation containingmultiple, different double stranded DNA molecules can be used astemplate DNA to amplify a locus or loci of interest contained in thetemplate DNA.

The term “amplification reaction” as used herein refers to a process forcopying nucleic acid one or more times. In embodiments, the method ofamplification includes but is not limited to polymerase chain reaction,self-sustained sequence reaction, ligase chain reaction, rapidamplification of cDNA ends, polymerase chain reaction and ligase chainreaction, O-beta phage amplification, strand displacement amplification,or splice overlap extension polymerase chain reaction. In someembodiments, a single molecule of nucleic acid is amplified, forexample, by digital PCR.

The term “sensitivity” as used herein refers to the number of truepositives divided by the number of true positives plus the number offalse negatives, where sensitivity (sens) may be within the range of0≦sens≦1. Ideally, method embodiments herein have the number of falsenegatives equaling zero or close to equaling zero, so that no subject iswrongly identified as not having at least one chromosome abnormality orother genetic disorder when they indeed have at least one chromosomeabnormality or other genetic disorder. Conversely, an assessment oftenis made of the ability of a prediction algorithm to classify negativescorrectly, a complementary measurement to sensitivity. The term“specificity” as used herein refers to the number of true negativesdivided by the number of true negatives plus the number of falsepositives, where sensitivity (spec) may be within the range of 0 spec 1.Ideally, methods embodiments herein have the number of false positivesequaling zero or close to equaling zero, so that no subject wronglyidentified as having at least one chromosome abnormality other geneticdisorder when they do not have the chromosome abnormality other geneticdisorder being assessed. Hence, a method that has sensitivity andspecificity equaling one, or 100%, sometimes is selected.

One or more prediction algorithms may be used to determine significanceor give meaning to the detection data collected under variableconditions that may be weighed independently of or dependently on eachother. The term “variable” as used herein refers to a factor, quantity,or function of an algorithm that has a value or set of values. Forexample, a variable may be the design of a set of amplified nucleic acidspecies, the number of sets of amplified nucleic acid species, percentfetal genetic contribution tested, percent maternal genetic contributiontested, type of chromosome abnormality assayed, type of genetic disorderassayed, type of sex-linked abnormalities assayed, the age of the motherand the like. The term “independent” as used herein refers to not beinginfluenced or not being controlled by another. The term “dependent” asused herein refers to being influenced or controlled by another. Forexample, a particular chromosome and a trisomy event occurring for thatparticular chromosome that results in a viable being are variables thatare dependent upon each other.

One of skill in the art may use any type of method or predictionalgorithm to give significance to the data of the present technologywithin an acceptable sensitivity and/or specificity. For example,prediction algorithms such as Chi-squared test, z-test, t-test, ANOVA(analysis of variance), regression analysis, neural nets, fuzzy logic,Hidden Markov Models, multiple model state estimation, and the like maybe used. One or more methods or prediction algorithms may be determinedto give significance to the data having different independent and/ordependent variables of the present technology. And one or more methodsor prediction algorithms may be determined not to give significance tothe data having different independent and/or dependent variables of thepresent technology. One may design or change parameters of the differentvariables of methods described herein based on results of one or moreprediction algorithms (e.g., number of sets analyzed, types ofnucleotide species in each set). For example, applying the Chi-squaredtest to detection data may suggest that specific ranges of maternal ageare correlated to a higher likelihood of having an offspring with aspecific chromosome abnormality, hence the variable of maternal age maybe weighed differently verses being weighed the same as other variables.

In certain embodiments, several algorithms may be chosen to be tested.These algorithms can be trained with raw data. For each new raw datasample, the trained algorithms will assign a classification to thatsample (i.e. trisomy or normal). Based on the classifications of the newraw data samples, the trained algorithms' performance may be assessedbased on sensitivity and specificity. Finally, an algorithm with thehighest sensitivity and/or specificity or combination thereof may beidentified.

DETAILED DESCRIPTION Introduction

The presence of fetal nucleic acid in maternal plasma was first reportedin 1997 and offers the possibility for non-invasive prenatal diagnosissimply through the analysis of a maternal blood sample (Lo et al.,Lancet 350:485-487, 1997). To date, numerous potential clinicalapplications have been developed. In particular, quantitativeabnormalities of fetal nucleic acid, for example DNA, concentrations inmaternal plasma have been found to be associated with a number ofpregnancy-associated disorders, including preeclampsia, preterm labor,antepartum hemorrhage, invasive placentation, fetal Down syndrome, andother fetal chromosomal aneuploidies. Hence, fetal nucleic acid analysisin maternal plasma represents a powerful mechanism for the monitoring offetomaternal well-being.

However, fetal DNA co-exists with background maternal DNA in maternalplasma. Hence, most reported applications have relied on the detectionof Y-chromosome sequences as these are most conveniently distinguishablefrom maternal DNA. Such an approach limits the applicability of theexisting assays to only 50% of all pregnancies, namely those with malefetuses. Thus, there is much need for the development of sex-independentcompositions and methods for enriching and analyzing fetal nucleic acidfrom a maternal sample. Also, methods that rely on polymorphic markersto quantify fetal nucleic acid may be susceptible to varyingheterozygosity rates across different ethnicities thereby limiting theirapplicability (e.g., by increasing the number of markers that areneeded).

It was previously demonstrated that fetal and maternal DNA can bedistinguished by their differences in methylation status (U.S. Pat. No.6,927,028, which is hereby incorporated by reference). Methylation is anepigenetic phenomenon, which refers to processes that alter a phenotypewithout involving changes in the DNA sequence. By exploiting thedifference in the DNA methylation status between mother and fetus, onecan successfully detect and analyze fetal nucleic acid in a backgroundof maternal nucleic acid.

The present inventors provides novel genomic polynucleotides that aredifferentially methylated between the fetal DNA from the fetus (e.g.,from the placenta) and the maternal DNA from the mother, for examplefrom peripheral blood cells. This discovery thus provides a new approachfor distinguishing fetal and maternal genomic DNA and new methods foraccurately quantifying fetal nucleic which may be used for non-invasiveprenatal diagnosis.

Methodology

Practicing the technology utilizes routine techniques in the field ofmolecular biology. Basic texts disclosing the general methods of use inthe technology include Sambrook and Russell, Molecular Cloning, ALaboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Protein sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized, e.g., according to the solid phase phosphoramidite triestermethod first described by Beaucage & Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), using an automated synthesizer, as described in VanDevanter et. al., Nucleic Acids Res. 12: 6159-6168 (1984). Purificationof oligonucleotides is performed using any art-recognized strategy,e.g., native acrylamide gel electrophoresis or anion-exchange highperformance liquid chromatography (HPLC) as described in Pearson &Reanier, J. Chrom. 255: 137-149 (1983).

Acquisition of Blood Samples and Extraction of DNA

The present technology relates to separating, enriching and analyzingfetal DNA found in maternal blood as a non-invasive means to detect thepresence and/or to monitor the progress of a pregnancy-associatedcondition or disorder. Thus, the first steps of practicing thetechnology are to obtain a blood sample from a pregnant woman andextract DNA from the sample.

A. Acquisition of Blood Samples

A blood sample is obtained from a pregnant woman at a gestational agesuitable for testing using a method of the present technology. Thesuitable gestational age may vary depending on the disorder tested, asdiscussed below. Collection of blood from a woman is performed inaccordance with the standard protocol hospitals or clinics generallyfollow. An appropriate amount of peripheral blood, e.g., typicallybetween 5-50 ml, is collected and may be stored according to standardprocedure prior to further preparation. Blood samples may be collected,stored or transported in a manner known to the person of ordinary skillin the art to minimize degradation or the quality of nucleic acidpresent in the sample.

B. Preparation of Blood Samples

The analysis of fetal DNA found in maternal blood according to thepresent technology may be performed using, e.g., the whole blood, serum,or plasma. The methods for preparing serum or plasma from maternal bloodare well known among those of skill in the art. For example, a pregnantwoman's blood can be placed in a tube containing EDTA or a specializedcommercial product such as Vacutainer SST (Becton Dickinson, FranklinLakes, N.J.) to prevent blood clotting, and plasma can then be obtainedfrom whole blood through centrifugation. On the other hand, serum may beobtained with or without centrifugation-following blood clotting. Ifcentrifugation is used then it is typically, though not exclusively,conducted at an appropriate speed, e.g., 1,500-3,000 times g. Plasma orserum may be subjected to additional centrifugation steps before beingtransferred to a fresh tube for DNA extraction.

In addition to the acellular portion of the whole blood, DNA may also berecovered from the cellular fraction, enriched in the buffy coatportion, which can be obtained following centrifugation of a whole bloodsample from the woman and removal of the plasma.

C. Extraction of DNA

There are numerous known methods for extracting DNA from a biologicalsample including blood. The general methods of DNA preparation (e.g.,described by Sambrook and Russell, Molecular Cloning: A LaboratoryManual 3d ed., 2001) can be followed; various commercially availablereagents or kits, such as Qiagen's QIAamp Circulating Nucleic Acid Kit,QiaAmp DNA Mini Kit or QiaAmp DNA Blood Mini Kit (Qiagen, Hilden,Germany), GenomicPrep™ Blood DNA Isolation Kit (Promega, Madison, Wis.),and GFX™ Genomic Blood DNA Purification Kit (Amersham, Piscataway,N.J.), may also be used to obtain DNA from a blood sample from apregnant woman. Combinations of more than one of these methods may alsobe used.

In some embodiments, the sample may first be enriched or relativelyenriched for fetal nucleic acid by one or more methods. For example, thediscrimination of fetal and maternal DNA can be performed using thecompositions and processes of the present technology alone or incombination with other discriminating factors. Examples of these factorsinclude, but are not limited to, single nucleotide differences betweenchromosome X and Y, chromosome Y-specific sequences, polymorphismslocated elsewhere in the genome, size differences between fetal andmaternal DNA and differences in methylation pattern between maternal andfetal tissues.

Other methods for enriching a sample for a particular species of nucleicacid are described in PCT Patent Application Number PCT/US07/69991,filed May 30, 2007, PCT Patent Application Number PCT/US2007/071232,filed Jun. 15, 2007, U.S. Provisional Application Nos. 60/968,876 and60/968,878 (assigned to the Applicant), (PCT Patent Application NumberPCT/EP05/012707, filed Nov. 28, 2005) which are all hereby incorporatedby reference. In certain embodiments, maternal nucleic acid isselectively removed (either partially, substantially, almost completelyor completely) from the sample.

Methylation Specific Separation of Nucleic Acid

The methods provided herein offer an alternative approach for theenrichment of fetal DNA based on the methylation-specific separation ofdifferentially methylated DNA. It has recently been discovered that manygenes involved in developmental regulation are controlled throughepigenetics in embryonic stem cells. Consequently, multiple genes can beexpected to show differential DNA methylation between nucleic acid offetal origin and maternal origin. Once these regions are identified, atechnique to capture methylated DNA can be used to specifically enrichfetal DNA. For identification of differentially methylated regions, anovel approach was used to capture methylated DNA. This approach uses aprotein, in which the methyl binding domain of MBD2 is fused to the Fcfragment of an antibody (MBD-FC) (Gebhard C, Schwarzfischer L, Pham T H,Schilling E, Klug M, Andreesen R, Rehli M (2006) Genomewide profiling ofCpG methylation identifies novel targets of aberrant hypermethylation inmyeloid leukemia. Cancer Res 66:6118-6128). This fusion protein hasseveral advantages over conventional methylation specific antibodies.The MBD-FC has a higher affinity to methylated DNA and it binds doublestranded DNA. Most importantly the two proteins differ in the way theybind DNA. Methylation specific antibodies bind DNA stochastically, whichmeans that only a binary answer can be obtained. The methyl bindingdomain of MBD-FC on the other hand binds DNA molecules regardless oftheir methylation status. The strength of this protein-DNA interactionis defined by the level of DNA methylation. After binding genomic DNA,eluate solutions of increasing salt concentrations can be used tofractionate non-methylated and methylated DNA allowing for a morecontrolled separation (Gebhard C, Schwarzfischer L, Pham T H, AndreesenR, Mackensen A, Rehli M (2006) Rapid and sensitive detection ofCpG-methylation using methyl-binding (MB)-PCR. Nucleic Acids Res34:e82). Consequently this method, called Methyl-CpG immunoprecipitation(MCIP), cannot only enrich, but also fractionate genomic DNA accordingto methylation level, which is particularly helpful when theunmethylated DNA fraction should be investigated as well.

Methylation Sensitive Restriction Enzyme Digestion

The technology also provides compositions and processes for determiningthe amount of fetal nucleic acid from a maternal sample. The technologyallows for the enrichment of fetal nucleic acid regions in a maternalsample by selectively digesting nucleic acid from said maternal samplewith an enzyme that selectively and completely or substantially digeststhe maternal nucleic acid to enrich the sample for at least one fetalnucleic acid region. Preferably, the digestion efficiency is greaterthan about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.Following enrichment, the amount of fetal nucleic acid can be determinedby quantitative methods that do not require polymorphic sequences orbisulfite treatment, thereby, offering a solution that works equallywell for female fetuses and across different ethnicities and preservesthe low copy number fetal nucleic acid present in the sample.

For example, there are methyl-sensitive enzymes that preferentially orsubstantially cleave or digest at their DNA recognition sequence if itis non-methylated. Thus, an unmethylated DNA sample will be cut intosmaller fragments than a methylated DNA sample. Similarly, ahypermethylated DNA sample will not be cleaved. In contrast, there aremethyl-sensitive enzymes that cleave at their DNA recognition sequenceonly if it is methylated.

Methyl-sensitive enzymes that digest unmethylated DNA suitable for usein methods of the technology include, but are not limited to, HpaII,HhaI, MaeII, BstUI and AciI. An enzyme that can be used is HpaII thatcuts only the unmethylated sequence CCGG. Another enzyme that can beused is HhaI that cuts only the unmethylated sequence GCGC. Both enzymesare available from New England BioLabs®, Inc. Combinations of two ormore methyl-sensitive enzymes that digest only unmethylated DNA can alsobe used. Suitable enzymes that digest only methylated DNA include, butare not limited to, DpnI, which cuts at a recognition sequence GATC, andMcrBC, which belongs to the family of AAA.sup.+ proteins and cuts DNAcontaining modified cytosines and cuts at recognition site 5′ . . .Pu.sup.mC(N.sub.40-3000) Pu.sup.mC . . . 3′ (New England BioLabs, Inc.,Beverly, Mass.).

Cleavage methods and procedures for selected restriction enzymes forcutting DNA at specific sites are well known to the skilled artisan. Forexample, many suppliers of restriction enzymes provide information onconditions and types of DNA sequences cut by specific restrictionenzymes, including New England BioLabs, Pro-Mega Biochems,Boehringer-Mannheim, and the like. Sambrook et al. (See Sambrook et al.,Molecular Biology: A laboratory Approach, Cold Spring Harbor, N.Y. 1989)provide a general description of methods for using restriction enzymesand other enzymes. In methods of the present technology enzymes oftenare used under conditions that will enable cleavage of the maternal DNAwith about 95%-100% efficiency, preferably with about 98%-100%efficiency.

Other Methods for Methylation Analysis

Various methylation analysis procedures are known in the art, and can beused in conjunction with the present technology. These assays allow fordetermination of the methylation state of one or a plurality of CpGislands within a DNA sequence. In addition, the methods maybe used toquantify methylated nucleic acid. Such assays involve, among othertechniques, DNA sequencing of bisulfite-treated DNA, PCR (forsequence-specific amplification), Southern blot analysis, and use ofmethylation-sensitive restriction enzymes.

Genomic sequencing is a technique that has been simplified for analysisof DNA methylation patterns and 5-methylcytosine distribution by usingbisulfite treatment (Frommer et al., Proc. Natl. Acad. Sci. USA89:1827-1831, 1992). Additionally, restriction enzyme digestion of PCRproducts amplified from bisulfite-converted DNA may be used, e.g., themethod described by Sadri & Hornsby (Nucl. Acids Res. 24:5058-5059,1996), or COBRA (Combined Bisulfite Restriction Analysis) (Xiong &Laird, Nucleic Acids Res. 25:2532-2534, 1997).

COBRA analysis is a quantitative methylation assay useful fordetermining DNA methylation levels at specific gene loci in smallamounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534,1997). Briefly, restriction enzyme digestion is used to revealmethylation-dependent sequence differences in PCR products of sodiumbisulfite-treated DNA. Methylation-dependent sequence differences arefirst introduced into the genomic DNA by standard bisulfite treatmentaccording to the procedure described by Frommer et al. (Proc. Natl.Acad. Sci. USA 89:1827-1831, 1992). PCR amplification of the bisulfiteconverted DNA is then performed using primers specific for theinterested CpG islands, followed by restriction endonuclease digestion,gel electrophoresis, and detection using specific, labeled hybridizationprobes. Methylation levels in the original DNA sample are represented bythe relative amounts of digested and undigested PCR product in alinearly quantitative fashion across a wide spectrum of DNA methylationlevels. In addition, this technique can be reliably applied to DNAobtained from microdissected paraffin-em bedded tissue samples. Typicalreagents (e.g., as might be found in a typical COBRA-based kit) forCOBRA analysis may include, but are not limited to: PCR primers forspecific gene (or methylation-altered DNA sequence or CpG island);restriction enzyme and appropriate buffer; gene-hybridization oligo;control hybridization oligo; kinase labeling kit for oligo probe; andradioactive nucleotides. Additionally, bisulfite conversion reagents mayinclude: DNA denaturation buffer; sulfonation buffer; DNA recoveryreagents or kits (e.g., precipitation, ultrafiltration, affinitycolumn); desulfonation buffer; and DNA recovery components.

The MethyLight™ assay is a high-throughput quantitative methylationassay that utilizes fluorescence-based real-time PCR (TaqMan®)technology that requires no further manipulations after the PCR step(Eads et al., Cancer Res. 59:2302-2306, 1999). Briefly, the MethyLight™process begins with a mixed sample of genomic DNA that is converted, ina sodium bisulfite reaction, to a mixed pool of methylation-dependentsequence differences according to standard procedures (the bisulfiteprocess converts unmethylated cytosine residues to uracil).Fluorescence-based PCR is then performed either in an “unbiased” (withprimers that do not overlap known CpG methylation sites) PCR reaction,or in a “biased” (with PCR primers that overlap known CpG dinucleotides)reaction. Sequence discrimination can occur either at the level of theamplification process or at the level of the fluorescence detectionprocess, or both.

The MethyLight assay may be used as a quantitative test for methylationpatterns in the genomic DNA sample, wherein sequence discriminationoccurs at the level of probe hybridization. In this quantitativeversion, the PCR reaction provides for unbiased amplification in thepresence of a fluorescent probe that overlaps a particular putativemethylation site. An unbiased control for the amount of input DNA isprovided by a reaction in which neither the primers, nor the probeoverlie any CpG dinucleotides. Alternatively, a qualitative test forgenomic methylation is achieved by probing of the biased PCR pool witheither control oligonucleotides that do not “cover” known methylationsites (a fluorescence-based version of the “MSP” technique), or witholigonucleotides covering potential methylation sites.

The MethyLight process can by used with a “TaqMan” probe in theamplification process. For example, double-stranded genomic DNA istreated with sodium bisulfite and subjected to one of two sets of PCRreactions using TaqMan® probes; e.g., with either biased primers andTaqMan® probe, or unbiased primers and TaqMan® probe. The TaqMan® probeis dual-labeled with fluorescent “reporter” and “quencher” molecules,and is designed to be specific for a relatively high GC content regionso that it melts out at about 10.degree. C. higher temperature in thePCR cycle than the forward or reverse primers. This allows the TaqMan®probe to remain fully hybridized during the PCR annealing/extensionstep. As the Taq polymerase enzymatically synthesizes a new strandduring PCR, it will eventually reach the annealed TaqMan® probe. The Taqpolymerase 5′ to 3′ endonuclease activity will then displace the TaqMan®probe by digesting it to release the fluorescent reporter molecule forquantitative detection of its now unquenched signal using a real-timefluorescent detection system.

Typical reagents (e.g., as might be found in a typical MethyLight™-basedkit) for MethyLight™ analysis may include, but are not limited to: PCRprimers for specific gene (or methylation-altered DNA sequence or CpGisland); TaqMan® probes; optimized PCR buffers and deoxynucleotides; andTaq polymerase.

The Ms-SNuPE technique is a quantitative method for assessingmethylation differences at specific CpG sites based on bisulfitetreatment of DNA, followed by single-nucleotide primer extension(Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997).

Briefly, genomic DNA is reacted with sodium bisulfite to convertunmethylated cytosine to uracil while leaving 5-methylcytosineunchanged. Amplification of the desired target sequence is thenperformed using PCR primers specific for bisulfite-converted DNA, andthe resulting product is isolated and used as a template for methylationanalysis at the CpG site(s) of interest.

Small amounts of DNA can be analyzed (e.g., microdissected pathologysections), and it avoids utilization of restriction enzymes fordetermining the methylation status at CpG sites.

Typical reagents (e.g., as might be found in a typical Ms-SNuPE-basedkit) for Ms-SNuPE analysis may include, but are not limited to: PCRprimers for specific gene (or methylation-altered DNA sequence or CpGisland); optimized PCR buffers and deoxynucleotides; gel extraction kit;positive control primers; Ms-SNuPE primers for specific gene; reactionbuffer (for the Ms-SNuPE reaction); and radioactive nucleotides.Additionally, bisulfite conversion reagents may include: DNAdenaturation buffer; sulfonation buffer; DNA recovery regents or kit(e.g., precipitation, ultrafiltration, affinity column); desulfonationbuffer; and DNA recovery components.

MSP (methylation-specific PCR) allows for assessing the methylationstatus of virtually any group of CpG sites within a CpG island,independent of the use of methylation-sensitive restriction enzymes(Herman et al. Proc. Nat. Acad. Sci. USA 93:9821-9826, 1996; U.S. Pat.No. 5,786,146). Briefly, DNA is modified by sodium bisulfite convertingunmethylated, but not methylated cytosines to uracil, and subsequentlyamplified with primers specific for methylated versus umethylated DNA.MSP requires only small quantities of DNA, is sensitive to 0.1%methylated alleles of a given CpG island locus, and can be performed onDNA extracted from paraffin-embedded samples. Typical reagents (e.g., asmight be found in a typical MSP-based kit) for MSP analysis may include,but are not limited to: methylated and unmethylated PCR primers forspecific gene (or methylation-altered DNA sequence or CpG island),optimized PCR buffers and deoxynucleotides, and specific probes.

The MCA technique is a method that can be used to screen for alteredmethylation patterns in genomic DNA, and to isolate specific sequencesassociated with these changes (Toyota et al., Cancer Res. 59:2307-12,1999). Briefly, restriction enzymes with different sensitivities tocytosine methylation in their recognition sites are used to digestgenomic DNAs from primary tumors, cell lines, and normal tissues priorto arbitrarily primed PCR amplification. Fragments that showdifferential methylation are cloned and sequenced after resolving thePCR products on high-resolution polyacrylamide gels. The clonedfragments are then used as probes for Southern analysis to confirmdifferential methylation of these regions. Typical reagents (e.g., asmight be found in a typical MCA-based kit) for MCA analysis may include,but are not limited to: PCR primers for arbitrary priming Genomic DNA;PCR buffers and nucleotides, restriction enzymes and appropriatebuffers; gene-hybridization oligos or probes; control hybridizationoligos or probes.

Another method for analyzing methylation sites is a primer extensionassay, including an optimized PCR amplification reaction that producesamplified targets for subsequent primer extension genotyping analysisusing mass spectrometry. The assay can also be done in multiplex. Thismethod (particularly as it relates to genotyping single nucleotidepolymorphisms) is described in detail in PCT publication WO05012578A1and US publication US20050079521A1. For methylation analysis, the assaycan be adopted to detect bisulfite introduced methylation dependent C toT sequence changes. These methods are particularly useful for performingmultiplexed amplification reactions and multiplexed primer extensionreactions (e.g., multiplexed homogeneous primer mass extension (hME)assays) in a single well to further increase the throughput and reducethe cost per reaction for primer extension reactions.

Four additional methods for DNA methylation analysis include restrictionlandmark genomic scanning (RLGS, Costello et al., 2000),methylation-sensitive-representational difference analysis (MS-RDA),methylation-specific AP-PCR (MS-AP-PCR) and methyl-CpG binding domaincolumn/segregation of partly melted molecules (MBD/SPM).

Additional methylation analysis methods that may be used in conjunctionwith the present technology are described in the following papers:Laird, P. W. Nature Reviews Cancer 3, 253-266 (2003); Biotechniques;Uhlmann, K. et al. Electrophoresis 23:4072-4079 (2002)—PyroMeth; Colellaet al. Biotechniques. 2003 July; 35(1):146-50; Dupont J M, Tost J,Jammes H, and Gut I G. Anal Biochem, October 2004; 333(1): 119-27; andTooke N and Pettersson M. IVDT. November 2004; 41.

Polynucleotide Sequence Amplification and Determination

Following separation of nucleic acid in a methylation-differentialmanner, the nucleic acid may be subjected to sequence-based analysis.Furthermore, once it is determined that one particular genomic sequenceof fetal origin is hypermethylated or hypomethylated compared to thematernal counterpart, the amount of this fetal genomic sequence can bedetermined. Subsequently, this amount can be compared to a standardcontrol value and serve as an indication for the potential of certainpregnancy-associated disorder.

A. Amplification of Nucleotide Sequences

In many instances, it is desirable to amplify a nucleic acid sequence ofthe technology using any of several nucleic acid amplificationprocedures which are well known in the art (listed above and describedin greater detail below). Specifically, nucleic acid amplification isthe enzymatic synthesis of nucleic acid amplicons (copies) which containa sequence that is complementary to a nucleic acid sequence beingamplified. Nucleic acid amplification is especially beneficial when theamount of target sequence present in a sample is very low. By amplifyingthe target sequences and detecting the amplicon synthesized, thesensitivity of an assay can be vastly improved, since fewer targetsequences are needed at the beginning of the assay to better ensuredetection of nucleic acid in the sample belonging to the organism orvirus of interest.

A variety of polynucleotide amplification methods are well establishedand frequently used in research. For instance, the general methods ofpolymerase chain reaction (PCR) for polynucleotide sequenceamplification are well known in the art and are thus not described indetail herein. For a review of PCR methods, protocols, and principles indesigning primers, see, e.g., Innis, et al., PCR Protocols: A Guide toMethods and Applications, Academic Press, Inc. N.Y., 1990. PCR reagentsand protocols are also available from commercial vendors, such as RocheMolecular Systems.

PCR is most usually carried out as an automated process with athermostable enzyme. In this process, the temperature of the reactionmixture is cycled through a denaturing region, a primer annealingregion, and an extension reaction region automatically. Machinesspecifically adapted for this purpose are commercially available.

Although PCR amplification of a polynucleotide sequence is typicallyused in practicing the present technology, one of skill in the art willrecognize that the amplification of a genomic sequence found in amaternal blood sample may be accomplished by any known method, such asligase chain reaction (LCR), transcription-mediated amplification, andself-sustained sequence replication or nucleic acid sequence-basedamplification (NASBA), each of which provides sufficient amplification.More recently developed branched-DNA technology may also be used toqualitatively demonstrate the presence of a particular genomic sequenceof the technology, which represents a particular methylation pattern, orto quantitatively determine the amount of this particular genomicsequence in the maternal blood. For a review of branched-DNA signalamplification for direct quantitation of nucleic acid sequences inclinical samples, see Nolte, Adv. Clin. Chem. 33:201-235, 1998.

The compositions and processes of the technology are also particularlyuseful when practiced with digital PCR. Digital PCR was first developedby Kalinina and colleagues (Kalinina et al., “Nanoliter scale PCR withTaqMan detection.” Nucleic Acids Research. 25; 1999-2004, (1997)) andfurther developed by Vogelstein and Kinzler (Digital PCR. Proc Natl AcadSci USA. 96; 9236-41, (1999)). The application of digital PCR for usewith fetal diagnostics was first described by Cantor et al. (PCT PatentPublication No. WO05023091A2) and subsequently described by Quake et al.(US Patent Publication No. US 20070202525), which are both herebyincorporated by reference. Digital PCR takes advantage of nucleic acid(DNA, cDNA or RNA) amplification on a single molecule level, and offersa highly sensitive method for quantifying low copy number nucleic acid.Fluidigm® Corporation offers systems for the digital analysis of nucleicacids.

B. Determination of Polynucleotide Sequences

Techniques for polynucleotide sequence determination are also wellestablished and widely practiced in the relevant research field. Forinstance, the basic principles and general techniques for polynucleotidesequencing are described in various research reports and treatises onmolecular biology and recombinant genetics, such as Wallace et al.,supra; Sambrook and Russell, supra, and Ausubel et al., supra. DNAsequencing methods routinely practiced in research laboratories, eithermanual or automated, can be used for practicing the present technology.Additional means suitable for detecting changes in a polynucleotidesequence for practicing the methods of the present technology includebut are not limited to mass spectrometry, primer extension,polynucleotide hybridization, real-time PCR, and electrophoresis.

Use of a primer extension reaction also can be applied in methods of thetechnology. A primer extension reaction operates, for example, bydiscriminating the SNP alleles by the incorporation of deoxynucleotidesand/or dideoxynucleotides to a primer extension primer which hybridizesto a region adjacent to the SNP site. The primer is extended with apolymerase. The primer extended SNP can be detected physically by massspectrometry or by a tagging moiety such as biotin. As the SNP site isonly extended by a complementary deoxynucleotide or dideoxynucleotidethat is either tagged by a specific label or generates a primerextension product with a specific mass, the SNP alleles can bediscriminated and quantified.

Reverse transcribed and amplified nucleic acids may be modified nucleicacids. Modified nucleic acids can include nucleotide analogs, and incertain embodiments include a detectable label and/or a capture agent.Examples of detectable labels include without limitation fluorophores,radioisotopes, colormetric agents, light emitting agents,chemiluminescent agents, light scattering agents, enzymes and the like.Examples of capture agents include without limitation an agent from abinding pair selected from antibody/antigen, antibody/antibody,antibody/antibody fragment, antibody/antibody receptor, antibody/proteinA or protein G, hapten/anti-hapten, biotin/avidin, biotin/streptavidin,folic acid/folate binding protein, vitamin B12/intrinsic factor,chemical reactive group/complementary chemical reactive group (e.g.,sulfhydryl/maleimide, sulfhydryl/haloacetyl derivative,amine/isotriocyanate, amine/succinimidyl ester, and amine/sulfonylhalides) pairs, and the like. Modified nucleic acids having a captureagent can be immobilized to a solid support in certain embodiments

Mass spectrometry is a particularly effective method for the detectionof a polynucleotide of the technology, for example a PCR amplicon, aprimer extension product or a detector probe that is cleaved from atarget nucleic acid. The presence of the polynucleotide sequence isverified by comparing the mass of the detected signal with the expectedmass of the polynucleotide of interest. The relative signal strength,e.g., mass peak on a spectra, for a particular polynucleotide sequenceindicates the relative population of a specific allele, thus enablingcalculation of the allele ratio directly from the data. For a review ofgenotyping methods using Sequenom® standard iPLEX™ assay and MassARRAY®technology, see Jurinke, C., Oeth, P., van den Boom, D., “MALDI-TOF massspectrometry: a versatile tool for high-performance DNA analysis.” Mol.Biotechnol. 26, 147-164 (2004); and Oeth, P. et al., “iPLEX™ Assay:Increased Plexing Efficiency and Flexibility for MassARRAY® Systemthrough single base primer extension with mass-modified Terminators.”SEQUENOM Application Note (2005), both of which are hereby incorporatedby reference. For a review of detecting and quantifying target nucleicusing cleavable detector probes that are cleaved during theamplification process and detected by mass spectrometry, see U.S. patentapplication Ser. No. 11/950,395, which was filed Dec. 4, 2007, and ishereby incorporated by reference.

Sequencing technologies are improving in terms of throughput and cost.Sequencing technologies, such as that achievable on the 454 platform(Roche) (Margulies, M. et al. 2005 Nature 437, 376-380), IIlumina GenomeAnalyzer (or Solexa platform) or SOLiD System (Applied Biosystems) orthe Helicos True Single Molecule DNA sequencing technology (Harris T Det al. 2008 Science, 320, 106-109), the single molecule, real-time(SMRT™) technology of Pacific Biosciences, and nanopore sequencing (SoniGV and Meller A. 2007 Clin Chem 53: 1996-2001), allow the sequencing ofmany nucleic acid molecules isolated from a specimen at high orders ofmultiplexing in a parallel fashion (Dear Brief Funct Genomic Proteomic2003; 1: 397-416).

Each of these platforms allow sequencing of clonally expanded ornon-amplified single molecules of nucleic acid fragments. Certainplatforms involve, for example, (i) sequencing by ligation ofdye-modified probes (including cyclic ligation and cleavage), (ii)pyrosequencing, and (iii) single-molecule sequencing. Nucleotidesequence species, amplification nucleic acid species and detectableproducts generated there from can be considered a “study nucleic acid”for purposes of analyzing a nucleotide sequence by such sequenceanalysis platforms.

Sequencing by ligation is a nucleic acid sequencing method that relieson the sensitivity of DNA ligase to base-pairing mismatch. DNA ligasejoins together ends of DNA that are correctly base paired. Combining theability of DNA ligase to join together only correctly base paired DNAends, with mixed pools of fluorescently labeled oligonucleotides orprimers, enables sequence determination by fluorescence detection.Longer sequence reads may be obtained by including primers containingcleavable linkages that can be cleaved after label identification.Cleavage at the linker removes the label and regenerates the 5′phosphate on the end of the ligated primer, preparing the primer foranother round of ligation. In some embodiments primers may be labeledwith more than one fluorescent label (e.g., 1 fluorescent label, 2, 3,or 4 fluorescent labels).

An example of a system that can be used by a person of ordinary skillbased on sequencing by ligation generally involves the following steps.Clonal bead populations can be prepared in emulsion microreactorscontaining study nucleic acid (“template”), amplification reactioncomponents, beads and primers. After amplification, templates aredenatured and bead enrichment is performed to separate beads withextended templates from undesired beads (e.g., beads with no extendedtemplates). The template on the selected beads undergoes a 3′modification to allow covalent bonding to the slide, and modified beadscan be deposited onto a glass slide. Deposition chambers offer theability to segment a slide into one, four or eight chambers during thebead loading process. For sequence analysis, primers hybridize to theadapter sequence. A set of four color dye-labeled probes competes forligation to the sequencing primer. Specificity of probe ligation isachieved by interrogating every 4th and 5th base during the ligationseries. Five to seven rounds of ligation, detection and cleavage recordthe color at every 5th position with the number of rounds determined bythe type of library used. Following each round of ligation, a newcomplimentary primer offset by one base in the 5′ direction is laid downfor another series of ligations. Primer reset and ligation rounds (5-7ligation cycles per round) are repeated sequentially five times togenerate 25-35 base pairs of sequence for a single tag. With mate-pairedsequencing, this process is repeated for a second tag. Such a system canbe used to exponentially amplify amplification products generated by aprocess described herein, e.g., by ligating a heterologous nucleic acidto the first amplification product generated by a process describedherein and performing emulsion amplification using the same or adifferent solid support originally used to generate the firstamplification product. Such a system also may be used to analyzeamplification products directly generated by a process described hereinby bypassing an exponential amplification process and directly sortingthe solid supports described herein on the glass slide.

Pyrosequencing is a nucleic acid sequencing method based on sequencingby synthesis, which relies on detection of a pyrophosphate released onnucleotide incorporation. Generally, sequencing by synthesis involvessynthesizing, one nucleotide at a time, a DNA strand complimentary tothe strand whose sequence is being sought. Study nucleic acids may beimmobilized to a solid support, hybridized with a sequencing primer,incubated with DNA polymerase, ATP sulfurylase, luciferase, apyrase,adenosine 5′ phosphsulfate and luciferin. Nucleotide solutions aresequentially added and removed. Correct incorporation of a nucleotidereleases a pyrophosphate, which interacts with ATP sulfurylase andproduces ATP in the presence of adenosine 5′ phosphsulfate, fueling theluciferin reaction, which produces a chemiluminescent signal allowingsequence determination.

An example of a system that can be used by a person of ordinary skillbased on pyrosequencing generally involves the following steps: ligatingan adaptor nucleic acid to a study nucleic acid and hybridizing thestudy nucleic acid to a bead; amplifying a nucleotide sequence in thestudy nucleic acid in an emulsion; sorting beads using a picolitermultiwell solid support; and sequencing amplified nucleotide sequencesby pyrosequencing methodology (e.g., Nakano et al., “Single-molecule PCRusing water-in-oil emulsion;” Journal of Biotechnology 102: 117-124(2003)). Such a system can be used to exponentially amplifyamplification products generated by a process described herein, e.g., byligating a heterologous nucleic acid to the first amplification productgenerated by a process described herein.

Certain single-molecule sequencing embodiments are based on theprincipal of sequencing by synthesis, and utilize single-pairFluorescence Resonance Energy Transfer (single pair FRET) as a mechanismby which photons are emitted as a result of successful nucleotideincorporation. The emitted photons often are detected using intensifiedor high sensitivity cooled charge-couple-devices in conjunction withtotal internal reflection microscopy (TIRM). Photons are only emittedwhen the introduced reaction solution contains the correct nucleotidefor incorporation into the growing nucleic acid chain that issynthesized as a result of the sequencing process. In FRET basedsingle-molecule sequencing, energy is transferred between twofluorescent dyes, sometimes polymethine cyanine dyes Cy3 and Cy5,through long-range dipole interactions. The donor is excited at itsspecific excitation wavelength and the excited state energy istransferred, non-radiatively to the acceptor dye, which in turn becomesexcited. The acceptor dye eventually returns to the ground state byradiative emission of a photon. The two dyes used in the energy transferprocess represent the “single pair”, in single pair FRET. Cy3 often isused as the donor fluorophore and often is incorporated as the firstlabeled nucleotide. Cy5 often is used as the acceptor fluorophore and isused as the nucleotide label for successive nucleotide additions afterincorporation of a first Cy3 labeled nucleotide. The fluorophoresgenerally are within 10 nanometers of each for energy transfer to occursuccessfully.

An example of a system that can be used based on single-moleculesequencing generally involves hybridizing a primer to a study nucleicacid to generate a complex; associating the complex with a solid phase;iteratively extending the primer by a nucleotide tagged with afluorescent molecule; and capturing an image of fluorescence resonanceenergy transfer signals after each iteration (e.g., U.S. Pat. No.7,169,314; Braslaysky et al., PNAS 100(7): 3960-3964 (2003)). Such asystem can be used to directly sequence amplification products generatedby processes described herein. In some embodiments the released linearamplification product can be hybridized to a primer that containssequences complementary to immobilized capture sequences present on asolid support, a bead or glass slide for example. Hybridization of theprimer—released linear amplification product complexes with theimmobilized capture sequences, immobilizes released linear amplificationproducts to solid supports for single pair FRET based sequencing bysynthesis. The primer often is fluorescent, so that an initial referenceimage of the surface of the slide with immobilized nucleic acids can begenerated. The initial reference image is useful for determininglocations at which true nucleotide incorporation is occurring.Fluorescence signals detected in array locations not initiallyidentified in the “primer only” reference image are discarded asnon-specific fluorescence. Following immobilization of theprimer—released linear amplification product complexes, the boundnucleic acids often are sequenced in parallel by the iterative steps of,a) polymerase extension in the presence of one fluorescently labelednucleotide, b) detection of fluorescence using appropriate microscopy,TIRM for example, c) removal of fluorescent nucleotide, and d) return tostep a with a different fluorescently labeled nucleotide.

In some embodiments, nucleotide sequencing may be by solid phase singlenucleotide sequencing methods and processes. Solid phase singlenucleotide sequencing methods involve contacting sample nucleic acid andsolid support under conditions in which a single molecule of samplenucleic acid hybridizes to a single molecule of a solid support. Suchconditions can include providing the solid support molecules and asingle molecule of sample nucleic acid in a “microreactor.” Suchconditions also can include providing a mixture in which the samplenucleic acid molecule can hybridize to solid phase nucleic acid on thesolid support. Single nucleotide sequencing methods useful in theembodiments described herein are described in U.S. Provisional PatentApplication Ser. No. 61/021,871 filed Jan. 17, 2008.

In certain embodiments, nanopore sequencing detection methods include(a) contacting a nucleic acid for sequencing (“base nucleic acid,” e.g.,linked probe molecule) with sequence-specific detectors, underconditions in which the detectors specifically hybridize tosubstantially complementary subsequences of the base nucleic acid; (b)detecting signals from the detectors and (c) determining the sequence ofthe base nucleic acid according to the signals detected. In certainembodiments, the detectors hybridized to the base nucleic acid aredisassociated from the base nucleic acid (e.g., sequentiallydissociated) when the detectors interfere with a nanopore structure asthe base nucleic acid passes through a pore, and the detectorsdisassociated from the base sequence are detected. In some embodiments,a detector disassociated from a base nucleic acid emits a detectablesignal, and the detector hybridized to the base nucleic acid emits adifferent detectable signal or no detectable signal. In certainembodiments, nucleotides in a nucleic acid (e.g., linked probe molecule)are substituted with specific nucleotide sequences corresponding tospecific nucleotides (“nucleotide representatives”), thereby giving riseto an expanded nucleic acid (e.g., U.S. Pat. No. 6,723,513), and thedetectors hybridize to the nucleotide representatives in the expandednucleic acid, which serves as a base nucleic acid. In such embodiments,nucleotide representatives may be arranged in a binary or higher orderarrangement (e.g., Soni and Meller, Clinical Chemistry 53(11): 1996-2001(2007)). In some embodiments, a nucleic acid is not expanded, does notgive rise to an expanded nucleic acid, and directly serves a basenucleic acid (e.g., a linked probe molecule serves as a non-expandedbase nucleic acid), and detectors are directly contacted with the basenucleic acid. For example, a first detector may hybridize to a firstsubsequence and a second detector may hybridize to a second subsequence,where the first detector and second detector each have detectable labelsthat can be distinguished from one another, and where the signals fromthe first detector and second detector can be distinguished from oneanother when the detectors are disassociated from the base nucleic acid.In certain embodiments, detectors include a region that hybridizes tothe base nucleic acid (e.g., two regions), which can be about 3 to about100 nucleotides in length (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80,85, 90, or 95 nucleotides in length). A detector also may include one ormore regions of nucleotides that do not hybridize to the base nucleicacid. In some embodiments, a detector is a molecular beacon. A detectoroften comprises one or more detectable labels independently selectedfrom those described herein. Each detectable label can be detected byany convenient detection process capable of detecting a signal generatedby each label (e.g., magnetic, electric, chemical, optical and thelike). For example, a CD camera can be used to detect signals from oneor more distinguishable quantum dots linked to a detector.

In certain sequence analysis embodiments, reads may be used to constructa larger nucleotide sequence, which can be facilitated by identifyingoverlapping sequences in different reads and by using identificationsequences in the reads. Such sequence analysis methods and software forconstructing larger sequences from reads are known to the person ofordinary skill (e.g., Venter et al., Science 291: 1304-1351 (2001)).Specific reads, partial nucleotide sequence constructs, and fullnucleotide sequence constructs may be compared between nucleotidesequences within a sample nucleic acid (i.e., internal comparison) ormay be compared with a reference sequence (i.e., reference comparison)in certain sequence analysis embodiments. Internal comparisons sometimesare performed in situations where a sample nucleic acid is prepared frommultiple samples or from a single sample source that contains sequencevariations. Reference comparisons sometimes are performed when areference nucleotide sequence is known and an objective is to determinewhether a sample nucleic acid contains a nucleotide sequence that issubstantially similar or the same, or different, than a referencenucleotide sequence. Sequence analysis is facilitated by sequenceanalysis apparatus and components known to the person of ordinary skillin the art.

Methods provided herein allow for high-throughput detection of nucleicacid species in a plurality of nucleic acids (e.g., nucleotide sequencespecies, amplified nucleic acid species and detectable productsgenerated from the foregoing). Multiplexing refers to the simultaneousdetection of more than one nucleic acid species. General methods forperforming multiplexed reactions in conjunction with mass spectrometry,are known (see, e.g., U.S. Pat. Nos. 6,043,031, 5,547,835 andInternational PCT application No. WO 97/37041). Multiplexing provides anadvantage that a plurality of nucleic acid species (e.g., some havingdifferent sequence variations) can be identified in as few as a singlemass spectrum, as compared to having to perform a separate massspectrometry analysis for each individual target nucleic acid species.Methods provided herein lend themselves to high-throughput,highly-automated processes for analyzing sequence variations with highspeed and accuracy, in some embodiments. In some embodiments, methodsherein may be multiplexed at high levels in a single reaction.

In certain embodiments, the number of nucleic acid species multiplexedinclude, without limitation, about 1 to about 500 (e.g., about 1-3, 3-5,5-7, 7-9, 9-11, 11-13, 13-15, 15-17, 17-19, 19-21, 21-23, 23-25, 25-27,27-29, 29-31, 31-33, 33-35, 35-37, 37-39, 39-41, 41-43, 43-45, 45-47,47-49, 49-51, 51-53, 53-55, 55-57, 57-59, 59-61, 61-63, 63-65, 65-67,67-69, 69-71, 71-73, 73-75, 75-77, 77-79, 79-81, 81-83, 83-85, 85-87,87-89, 89-91, 91-93, 93-95, 95-97, 97-101, 101-103, 103-105, 105-107,107-109, 109-111, 111-113, 113-115, 115-117, 117-119, 121-123, 123-125,125-127, 127-129, 129-131, 131-133, 133-135, 135-137, 137-139, 139-141,141-143, 143-145, 145-147, 147-149, 149-151, 151-153, 153-155, 155-157,157-159, 159-161, 161-163, 163-165, 165-167, 167-169, 169-171, 171-173,173-175, 175-177, 177-179, 179-181, 181-183, 183-185, 185-187, 187-189,189-191, 191-193, 193-195, 195-197, 197-199, 199-201, 201-203, 203-205,205-207, 207-209, 209-211, 211-213, 213-215, 215-217, 217-219, 219-221,221-223, 223-225, 225-227, 227-229, 229-231, 231-233, 233-235, 235-237,237-239, 239-241, 241-243, 243-245, 245-247, 247-249, 249-251, 251-253,253-255, 255-257, 257-259, 259-261, 261-263, 263-265, 265-267, 267-269,269-271, 271-273, 273-275, 275-277, 277-279, 279-281, 281-283, 283-285,285-287, 287-289, 289-291, 291-293, 293-295, 295-297, 297-299, 299-301,301-303, 303-305, 305-307, 307-309, 309-311, 311-313, 313-315, 315-317,317-319, 319-321, 321-323, 323-325, 325-327, 327-329, 329-331, 331-333,333-335, 335-337, 337-339, 339-341, 341-343, 343-345, 345-347, 347-349,349-351, 351-353, 353-355, 355-357, 357-359, 359-361, 361-363, 363-365,365-367, 367-369, 369-371, 371-373, 373-375, 375-377, 377-379, 379-381,381-383, 383-385, 385-387, 387-389, 389-391, 391-393, 393-395, 395-397,397-401, 401-403, 403-405, 405-407, 407-409, 409-411, 411-413, 413-415,415-417, 417-419, 419-421, 421-423, 423-425, 425-427, 427-429, 429-431,431-433, 433-435, 435-437, 437-439, 439-441, 441-443, 443-445, 445-447,447-449, 449-451, 451-453, 453-455, 455-457, 457-459, 459-461, 461-463,463-465, 465-467, 467-469, 469-471, 471-473, 473-475, 475-477, 477-479,479-481, 481-483, 483-485, 485-487, 487-489, 489-491, 491-493, 493-495,495-497, 497-501).

Design methods for achieving resolved mass spectra with multiplexedassays can include primer and oligonucleotide design methods andreaction design methods. See, for example, the multiplex schemesprovided in Tables X and Y. For primer and oligonucleotide design inmultiplexed assays, the same general guidelines for primer designapplies for uniplexed reactions, such as avoiding false priming andprimer dimers, only more primers are involved for multiplex reactions.For mass spectrometry applications, analyte peaks in the mass spectrafor one assay are sufficiently resolved from a product of any assay withwhich that assay is multiplexed, including pausing peaks and any otherby-product peaks. Also, analyte peaks optimally fall within auser-specified mass window, for example, within a range of 5,000-8,500Da. In some embodiments multiplex analysis may be adapted to massspectrometric detection of chromosome abnormalities, for example. Incertain embodiments multiplex analysis may be adapted to various singlenucleotide or nanopore based sequencing methods described herein.Commercially produced micro-reaction chambers or devices or arrays orchips may be used to facilitate multiplex analysis, and are commerciallyavailable.

Detection of Fetal Aneuploidy

For the detection of fetal aneuploidies, some methods rely on measuringthe ratio between maternally and paternally inherited alleles. However,the ability to quantify chromosomal changes is impaired by the maternalcontribution of cell free nucleic acids, which makes it necessary todeplete the sample from maternal DNA prior to measurement. Promisingapproaches take advantage of the different size distribution of fetaland maternal DNA or measure RNA that is exclusively expressed by thefetus (see for example, U.S. patent application Ser. No. 11/384,128,which published as US20060252071 and is hereby incorporated byreference). Assuming fetal DNA makes up only about 5% of all cell freeDNA in the maternal plasma, there is a decrease of the ratio differencefrom 1.6% to only about 1.2% between a trisomy sample and a healthycontrol. Consequently, reliable detection of allele ratio changesrequires enriching the fetal fraction of cell free DNA, for example,using the compositions and methods of the present technology.

Some methods rely on measuring the ratio of maternal to paternallyinherited alleles to detect fetal chromosomal aneuploidies from maternalplasma. A diploid set yields a 1:1 ratio while trisomies can be detectedas a 2:1 ratio. Detection of this difference is impaired by statisticalsampling due to the low abundance of fetal DNA, presence of excessmaternal DNA in the plasma sample and variability of the measurementtechnique. The latter is addressed by using methods with highmeasurement precision, like digital PCR or mass spectrometry. Enrichingthe fetal fraction of cell free DNA in a sample is currently achieved byeither depleting maternal DNA through size exclusion or focusing onfetal-specific nucleic acids, like fetal-expressed RNA. Anotherdifferentiating feature of fetal DNA is its DNA methylation pattern.Thus, provided herein are novel compositions and methods for accuratelyquantifying fetal nucleic acid based on differential methylation betweena fetus and mother. The methods rely on sensitive absolute copy numberanalysis to quantify the fetal nucleic acid portion of a maternalsample, thereby allowing for the prenatal detection of fetal traits. Themethods of the technology have identified approximately 3000 CpG richregions in the genome that are differentially methylated betweenmaternal and fetal DNA. The selected regions showed highly conserveddifferential methylation across all measured samples. In addition theset of regions is enriched for genes important in developmentalregulation, indicating that epigenetic regulation of these areas is abiologically relevant and consistent process (see Table 3). Enrichmentof fetal DNA can now be achieved by using our MBD-FC protein to capturecell free DNA (e.g., substantially all cell free DNA) and then elute thehighly methylated DNA fraction with high salt concentrations. Using thelow salt eluate fractions, the MBD-FC is equally capable of enrichingnon-methylated fetal DNA.

The present technology provides 63 confirmed genomic regions onchromosomes 13, 18 and 21 with low maternal and high fetal methylationlevels. After capturing these regions, SNPs can be used to determine theaforementioned allele ratios. When high frequency SNPs are used around10 markers have to be measured to achieve a high confidence of findingat least one SNP where the parents have opposite homozygote genotypesand the child has a heterozygote genotype.

In another embodiment, a method for chromosomal abnormality detection isprovided that utilizes absolute copy number quantification. A diploidchromosome set will show the same number of copies for differentiallymethylated regions across all chromosomes, but, for example, a trisomy21 sample would show 1.5 times more copies for differentially methylatedregions on chromosome 21. Normalization of the genomic DNA amounts for adiploid chromosome set can be achieved by using unaltered autosomes asreference (also provided herein—see Table 1). Comparable to otherapproaches, a single marker is less likely to be sufficient fordetection of this difference, because the overall copy numbers are low.Typically there are approximately 100 to 200 copies of fetal DNA from 1ml of maternal plasma at 10 to 12 weeks of gestation. However, themethods of the present technology offer a redundancy of detectablemarkers that enables highly reliable discrimination of diploid versusaneuploid chromosome sets.

Data Processing and Identifying Presence or Absence of a ChromosomeAbnormality

The term “detection” of a chromosome abnormality as used herein refersto identification of an imbalance of chromosomes by processing dataarising from detecting sets of amplified nucleic acid species,nucleotide sequence species, or a detectable product generated from theforegoing (collectively “detectable product”). Any suitable detectiondevice and method can be used to distinguish one or more sets ofdetectable products, as addressed herein. An outcome pertaining to thepresence or absence of a chromosome abnormality can be expressed in anysuitable form, including, without limitation, probability (e.g., oddsratio, p-value), likelihood, percentage, value over a threshold, or riskfactor, associated with the presence of a chromosome abnormality for asubject or sample. An outcome may be provided with one or more ofsensitivity, specificity, standard deviation, coefficient of variation(CV) and/or confidence level, or combinations of the foregoing, incertain embodiments.

Detection of a chromosome abnormality based on one or more sets ofdetectable products may be identified based on one or more calculatedvariables, including, but not limited to, sensitivity, specificity,standard deviation, coefficient of variation (CV), a threshold,confidence level, score, probability and/or a combination thereof. Insome embodiments, (i) the number of sets selected for a diagnosticmethod, and/or (ii) the particular nucleotide sequence species of eachset selected for a diagnostic method, is determined in part or in fullaccording to one or more of such calculated variables.

In certain embodiments, one or more of sensitivity, specificity and/orconfidence level are expressed as a percentage. In some embodiments, thepercentage, independently for each variable, is greater than about 90%(e.g., about 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, or greater than99% (e.g., about 99.5%, or greater, about 99.9% or greater, about 99.95%or greater, about 99.99% or greater)). Coefficient of variation (CV) insome embodiments is expressed as a percentage, and sometimes thepercentage is about 10% or less (e.g., about 10, 9, 8, 7, 6, 5, 4, 3, 2or 1%, or less than 1% (e.g., about 0.5% or less, about 0.1% or less,about 0.05% or less, about 0.01% or less)). A probability (e.g., that aparticular outcome determined by an algorithm is not due to chance) incertain embodiments is expressed as a p-value, and sometimes the p-valueis about 0.05 or less (e.g., about 0.05, 0.04, 0.03, 0.02 or 0.01, orless than 0.01 (e.g., about 0.001 or less, about 0.0001 or less, about0.00001 or less, about 0.000001 or less)).

For example, scoring or a score may refer to calculating the probabilitythat a particular chromosome abnormality is actually present or absentin a subject/sample. The value of a score may be used to determine forexample the variation, difference, or ratio of amplified nucleicdetectable product that may correspond to the actual chromosomeabnormality. For example, calculating a positive score from detectableproducts can lead to an identification of a chromosome abnormality,which is particularly relevant to analysis of single samples.

In certain embodiments, simulated (or simulation) data can aid dataprocessing for example by training an algorithm or testing an algorithm.Simulated data may for instance involve hypothetical various samples ofdifferent concentrations of fetal and maternal nucleic acid in serum,plasma and the like. Simulated data may be based on what might beexpected from a real population or may be skewed to test an algorithmand/or to assign a correct classification based on a simulated data set.Simulated data also is referred to herein as “virtual” data.Fetal/maternal contributions within a sample can be simulated as a tableor array of numbers (for example, as a list of peaks corresponding tothe mass signals of cleavage products of a reference biomolecule oramplified nucleic acid sequence), as a mass spectrum, as a pattern ofbands on a gel, or as a representation of any technique that measuresmass distribution. Simulations can be performed in most instances by acomputer program. One possible step in using a simulated data set is toevaluate the confidence of the identified results, i.e. how well theselected positives/negatives match the sample and whether there areadditional variations. A common approach is to calculate the probabilityvalue (p-value) which estimates the probability of a random samplehaving better score than the selected one. As p-value calculations canbe prohibitive in certain circumstances, an empirical model may beassessed, in which it is assumed that at least one sample matches areference sample (with or without resolved variations). Alternativelyother distributions such as Poisson distribution can be used to describethe probability distribution.

In certain embodiments, an algorithm can assign a confidence value tothe true positives, true negatives, false positives and false negativescalculated. The assignment of a likelihood of the occurrence of achromosome abnormality can also be based on a certain probability model.

Simulated data often is generated in an in silico process. As usedherein, the term “in silico” refers to research and experimentsperformed using a computer. In silico methods include, but are notlimited to, molecular modeling studies, karyotyping, geneticcalculations, biomolecular docking experiments, and virtualrepresentations of molecular structures and/or processes, such asmolecular interactions.

As used herein, a “data processing routine” refers to a process, thatcan be embodied in software, that determines the biological significanceof acquired data (i.e., the ultimate results of an assay). For example,a data processing routine can determine the amount of each nucleotidesequence species based upon the data collected. A data processingroutine also may control an instrument and/or a data collection routinebased upon results determined. A data processing routine and a datacollection routine often are integrated and provide feedback to operatedata acquisition by the instrument, and hence provide assay-basedjudging methods provided herein.

As used herein, software refers to computer readable programinstructions that, when executed by a computer, perform computeroperations. Typically, software is provided on a program productcontaining program instructions recorded on a computer readable medium,including, but not limited to, magnetic media including floppy disks,hard disks, and magnetic tape; and optical media including CD-ROM discs,DVD discs, magneto-optical discs, and other such media on which theprogram instructions can be recorded.

Different methods of predicting abnormality or normality can producedifferent types of results. For any given prediction, there are fourpossible types of outcomes: true positive, true negative, falsepositive, or false negative. The term “true positive” as used hereinrefers to a subject correctly diagnosed as having a chromosomeabnormality. The term “false positive” as used herein refers to asubject wrongly identified as having a chromosome abnormality. The term“true negative” as used herein refers to a subject correctly identifiedas not having a chromosome abnormality. The term “false negative” asused herein refers to a subject wrongly identified as not having achromosome abnormality. Two measures of performance for any given methodcan be calculated based on the ratios of these occurrences: (i) asensitivity value, the fraction of predicted positives that arecorrectly identified as being positives (e.g., the fraction ofnucleotide sequence sets correctly identified by level comparisondetection/determination as indicative of chromosome abnormality,relative to all nucleotide sequence sets identified as such, correctlyor incorrectly), thereby reflecting the accuracy of the results indetecting the chromosome abnormality; and (ii) a specificity value, thefraction of predicted negatives correctly identified as being negative(the fraction of nucleotide sequence sets correctly identified by levelcomparison detection/determination as indicative of chromosomalnormality, relative to all nucleotide sequence sets identified as such,correctly or incorrectly), thereby reflecting accuracy of the results indetecting the chromosome abnormality.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially the same or similar results.

In Example 1 below, the Applicants used a new fusion protein thatcaptures methylated DNA in combination with CpG Island array to identifygenomic regions that are differentially methylated between fetalplacenta tissue and maternal blood. A stringent statistical approach wasused to only select regions which show little variation between thesamples, and hence suggest an underlying biological mechanism.Eighty-five differentially methylated genomic regions predominantlylocated on chromosomes 13, 18 and 21 were validated. For thisvalidation, a quantitative mass spectrometry based approach was usedthat interrogated 261 PCR amplicons covering these 85 regions. Theresults are in very good concordance (95% confirmation), proving thefeasibility of the approach.

Next, the Applicants provide an innovative approach for aneuploidytesting, which relies on the measurement of absolute copy numbers ratherthan allele ratios.

Example 1

In the below Example, ten paired maternal and placental DNA samples wereused to identify differentially methylated regions. These results werevalidated using a mass spectrometry-based quantitative methylationassay. First, genomic DNA from maternal buffy coat and correspondingplacental tissue was first extracted. Next the MBD-FC was used tocapture the methylated fraction of each DNA sample. See FIGS. 1-3. Thetwo tissue fractions were labeled with different fluorescent dyes andhybridized to an Agilent® CpG Island microarray. See FIG. 4. This wasdone to identify differentially methylated regions that could beutilized for prenatal diagnoses. Therefore, two criteria were employedto select genomic regions as potential enrichment markers: the observedmethylation difference had to be present in all tested sample pairs, andthe region had to be more than 200 bp in length.

DNA Preparation and Fragmentation

Genomic DNA (gDNA) from maternal buffy coat and placental tissue wasprepared using the QIAamp DNA Mini Kit™ and QIAamp DNA Blood Mini Kit™,respectively, from Qiagen® (Hilden, Germany). For MCIp, gDNA wasquantified using the NanoDrop ND 1000™ spectrophotometer (ThermoFisher®, Waltham, Mass., USA). Ultrasonication of 2.5 μg DNA in 500 μlTE buffer to a mean fragment size of 300-500 bp was carried out with theBranson Digital Sonifier 450™ (Danbury, Conn., USA) using the followingsettings: amplitude 20%, sonication time 110 seconds, pulse on/pulse offtime 1.4/0.6 seconds. Fragment range was monitored using gelelectrophoresis.

Methyl-CpG Immunoprecipitation

Per sample, 56 μg purified MBD-Fc protein and 150 μl of Protein ASepharose 4 Fast Flow beads (Amersham Biosciences®, Piscataway, N.J.,USA) were rotated in 15 ml TBS overnight at 4° C. Then, the MBD-Fc beads(150 μl/assay) were transferred and dispersed in to 2 ml Ultrafree-CLcentrifugal filter devices (Millipore®, Billerica, Mass., USA) andspin-washed three times with Buffer A (20 mM Tris-HCl, pH8.0, 2 mMMgCl2, 0.5 mM EDTA 300 mM NaCl, 0.1% NP-40). Sonicated DNA (2 μg) wasadded to the washed MBD-Fc beads in 2 ml Buffer A and rotated for 3hours at 4° C. Beads were centrifuged to recover unbound DNA fragments(300 mM fraction) and subsequently washed twice with 600 μl of bufferscontaining increasing NaCl concentrations (400, 500, 550, 600, and 1000mM). The flow through of each wash step was collected in separate tubesand desalted using a MinElute PCR Purification Kit™ (Qiagen®). Inparallel, 200 ng sonicated input DNA was processed as a control usingthe MinElute PCR Purification Kit™ (Qiagen®). Microarray handling andanalysis

To generate fluorescently labeled DNA for microarray hybridization, the600 mM and 1M NaCl fractions (enriched methylated DNA) for each samplewere combined and labeled with either Alexa Fluor 555-aha-dCTP(maternal) or Alexa Fluor 647-aha-dCTP (placental) using the BioPrimeTotal Genomic Labeling System™ (Invitrogen®, Carlsbad, Calif., USA). Thelabeling reaction was carried out according to the manufacturer'smanual. The differently labeled genomic DNA fragments of matchedmaternal/placental pairs were combined to a final volume of 80 μl,supplemented with 50 μg Cot-1 DNA) (Invitrogen®, 52 μl of Agilent 10×blocking reagent (Agilent Technologies®, Santa Clara, Calif., USA), 78μl of deionized formamide, and 260 μl Agilent 2× hybridization buffer.The samples were heated to 95° C. for 3 min, mixed, and subsequentlyincubated at 37° C. for 30 min. Hybridization on Agilent CpG IslandMicroarray Kit™ was then carried out at 67° C. for 40 hours using anAgilent SureHyb™ chamber and an Agilent hybridization oven. Slides werewashed in Wash I (6×SSPE, 0.005% N-lauroylsarcosine) at room temperaturefor 5 min and in Wash II (0.06×SSPE) at 37° C. for an additional 5 min.Next, the slides were submerged in acetonitrile and Agilent OzoneProtection Solution™, respectively, for 30 seconds. Images were scannedimmediately and analyzed using an Agilent DNA Microarray Scanner™.Microarray images were processed using Feature Extraction Software v9.5and the standard CGH protocol.

Bisulfite Treatment

Genomic DNA sodium bisulfite conversion was performed using EZ-96 DNAMethylation Kit™ (ZymoResearch, Orange County, Calif.). Themanufacturer's protocol was followed using 1 ug of genomic DNA and thealternative conversion protocol (a two temperature DNA denaturation).

Quantitative Methylation Analysis

Sequenom's MassARRAY® System was used to perform quantitativemethylation analysis. This system utilizes matrix-assisted laserdesorption ionization time-of-flight (MALDI-TOF) mass spectrometry incombination with RNA base specific cleavage (Sequenom® MassCLEAVE™). Adetectable pattern is then analyzed for methylation status. PCR primerswere designed using Sequenom® EpiDESIGNER™ (www.epidesigner.com). Atotal of 261 amplicons, covering 85 target regions, were used forvalidation (median amplification length=367 bp, min=108, max=500; mediannumber of CpG's per amplicon=23, min=4, max=65). For each reverseprimer, an additional T7 promoter tag for in-vivo transcription wasadded, as well as a 10 mer tag on the forward primer to adjust formelting temperature differences. The MassCLEAVE™ biochemistry wasperformed as previously described (Ehrich M, et al. (2005) Quantitativehigh-throughput analysis of DNA methylation patterns by base specificcleavage and mass spectrometry. Proc Natl Acad Sci USA 102:15785-15790).Mass spectra were acquired using a MassARRAY™ Compact MALDI-TOF(Sequenom®, San Diego) and methylation ratios were generated by theEpiTYPER™ software v1.0 (Sequenom®, San Diego).

Statistical Analysis

All statistical calculations were performed using the R statisticalsoftware package (www.r-project.org). First, the array probes weregrouped based on their genomic location. Subsequent probes that wereless than 1000 bp apart were grouped together. To identifydifferentially methylated regions, a control sample was used asreference. In the control sample, the methylated fraction of a bloodderived control DNA was hybridized against itself. Ideally this sampleshould show log ratios of the two color channels around 0. Howeverbecause of the variability in hybridization behavior, the probes show amean log ratio of 0.02 and a standard deviation of 0.18. Next the logratios observed in our samples were compared to the control sample. Atwo way, paired t-test was used to test the NULL hypothesis that thegroups are identical. Groups that contained less than 4 probes wereexcluded from the analysis. For groups including four or five probes,all probes were used in a paired t-test. For Groups with six or moreprobes, a sliding window test consisting of five probes at a time wasused, whereby the window was moved by one probe increments. Each testsample was compared to the control sample and the p-values wererecorded. Genomic regions were selected as being differentiallymethylated if eight out of ten samples showed a p value<0.01, or if sixout of ten samples showed a p value<0.001. The genomic regions wereclassified as being not differentially methylated when the group showedless than eight samples with a p value<0.01 and less than six sampleswith a p value<0.001. Samples that didn't fall in either category wereexcluded from the analysis. For a subset of genomic regions that havebeen identified as differentially methylated, the results were confirmedusing quantitative methylation analysis.

The Go analysis was performed using the online GOstat tool(http://gostat.wehi.edu.au/cgibin/-goStat.pl). P values were calculatedusing Fisher's exact test.

Microarray-Based Marker Discovery Results

To identify differentially methylated regions a standard sample wasused, in which the methylated DNA fraction of monocytes was hybridizedagainst itself. This standard provided a reference for the variabilityof fluorescent measurements in a genomic region. Differentiallymethylated regions were then identified by comparing the log ratios ofeach of the ten placental/maternal samples against this standard.Because the goal of this study was to identify markers that allow thereliable separation of maternal and fetal DNA, the target selection waslimited to genes that showed a stable, consistent methylation differenceover a contiguous stretch of genomic DNA. This focused the analysis ongenomic regions where multiple probes indicated differentialmethylation. The selection was also limited to target regions where allsamples showed differential methylation, excluding those with stronginter-individual differences. Two of the samples showed generally lowerlog ratios in the microarray analysis. Because a paired test was usedfor target selection, this did not negatively impact the results.

Based on these selection criteria, 3043 genomic regions were identifiedthat were differentially methylated between maternal and fetal DNA.21778 regions did not show a methylation difference. Nointer-chromosomal bias in the distribution of differentially methylatedregions was observed. The differentially methylated regions were locatednext to or within 2159 known genes. The majority of differentiallymethylated regions are located in the promoter area (18%) and inside thecoding region (68%), while only few regions are located downstream ofthe gene (7%) or at the transition from promoter to coding region (7%).Regions that showed no differential methylation showed a similardistribution for promoter (13%) and downstream (5%) locations, but thefraction of regions located in the transition of promoter to codingregion was higher (39%) and the fraction inside the coding region waslower (43%).

It has been shown in embryonic stem cells (ES) that genes targeted bythe polycomb repressive complex2 (PRC2) are enriched for genesregulating development (Lee T I, et al. (2006) Control of developmentalregulators by Polycomb in human embryonic stem cells. Cell 125:301-313).It has also been shown that differentially methylated genes are enrichedfor genes targeted by PRC2 in many cancer types (Ehrich M, et al. (2008)Cytosine methylation profiling of cancer cell lines. Proc Natl Acad SciUSA 105:4844-48). The set of genes identified as differentiallymethylated in this study is also enriched for genes targeted by PRC2(p-value<0.001, odds ratio=3.6, 95% CI for odds ratio=3.1-4.2). A GOanalysis of the set of differentially methylated genes reveals that thisset is significantly enriched for functions important duringdevelopment. Six out of the ten most enriched functions includedevelopmental or morphogenic processes [anatomical structuremorphogenesis (GO:0009653, p value=0), developmental process(GO:0032502, p value=0), multicellular organismal development(GO:0007275, p value=0), developmental of an organ (GO:0048513, pvalue=0), system development (GO:0048731, p value=0) and development ofan anatomical structure (GO:0048856, p value=0)].

Validation Using Sequenom® EpiTYPER™

To validate the microarray findings, 63 regions from chromosomes 13, 18and 21 and an additional 26 regions from other autosomes were selectedfor confirmation by a different technology. Sequenom EpiTYPER™technology was used to quantitatively measure DNA methylation inmaternal and placental samples. For an explanation of the EpiTYPER™methods, see Ehrich M, Nelson M R, Stanssens P, Zabeau M, Liloglou T,Xinarianos G, Cantor C R, Field J K, van den Boom D (2005) Quantitativehigh-throughput analysis of DNA methylation patterns by base specificcleavage and mass spectrometry. Proc Natl Acad Sci USA 102:15785-15790).For each individual CpG site in a target region the average methylationvalue across all maternal DNA samples and across all placenta sampleswas calculated. The difference between average maternal and placentamethylation was then compared to the microarray results. The resultsfrom the two technologies were in good concordance (see FIG. 7). For 85target regions the quantitative results confirm the microarray results(95% confirmation rate). For 4 target regions, all located on chromosome18, the results could not be confirmed. The reason for this discrepancyis currently unclear.

In contrast to microarrays, which focus on identification of methylationdifferences, the quantitative measurement of DNA methylation allowedanalysis of absolute methylation values. In the validation set of 85confirmed differentially methylated regions, a subset of 26 regions ismore methylated in the maternal DNA sample and 59 regions are moremethylated in the placental sample (see Table 1). Interestingly, genesthat are hypomethylated in the placental samples tend to show largermethylation differences than genes that are hypermethylated in theplacental sample (median methylation difference for hypomethylatedgenes=39%, for hypermethylated genes=20%).

Example 2

Example 2 describes a non-invasive approach for detecting the amount offetal nucleic acid present in a maternal sample (herein referred to asthe “Fetal Quantifier Method”), which may be used to detect or confirmfetal traits (e.g., fetal sex of RhD compatibility), or diagnosechromosomal abnormalities such as Trisomy 21 (both of which are hereinreferred to as the “Methylation-Based Fetal Diagnostic Method”). FIG. 10shows one embodiment of the Fetal Quantifier Method, and FIG. 11 showsone embodiment of the Methylation-Based Fetal Diagnostic Method. Bothprocesses use fetal DNA obtained from a maternal sample. The samplecomprises maternal and fetal nucleic acid that is differentiallymethylated. For example, the sample may be maternal plasma or serum.Fetal DNA comprises approximately 2-30% of the total DNA in maternalplasma. The actual amount of fetal contribution to the total nucleicacid present in a sample varies from pregnancy to pregnancy and canchange based on a number of factors, including, but not limited to,gestational age, the mother's health and the fetus' health.

As described herein, the technical challenge posed by analysis of fetalDNA in maternal plasma lies in the need to be able to discriminate thefetal DNA from the co-existing background maternal DNA. The methods ofthe present technology exploit such differences, for example, thedifferential methylation that is observed between fetal and maternalDNA, as a means to enrich for the relatively small percentage of fetalDNA present in a sample from the mother. The non-invasive nature of theapproach provides a major advantage over conventional methods ofprenatal diagnosis such as, amniocentesis, chronic villus sampling andcordocentesis, which are associated with a small but finite risk offetal loss. Also, because the method is not dependent on fetal cellsbeing in any particular cell phase, the method provides a rapiddetection means to determine the presence and also the nature of thechromosomal abnormality. Further, the approach is sex-independent (i.e.,does not require the presence of a Y-chromosome) andpolymorphic-independent (i.e., an allelic ratio is not determined).Thus, the compositions and methods of the technology represent improveduniversal, noninvasive approaches for accurately determining the amountof fetal nucleic acid present in a maternal sample.

Assay Design and Advantages

There is a need for accurate detection and quantification of fetal DNAisolated noninvasively from a maternal sample. The present technologytakes advantage of the presence of circulating, cell free fetal nucleicacid (ccfDNA) in maternal plasma or serum. In order to be commerciallyand clinically practical, the methods of the technology should onlyconsume a small portion of the limited available fetal DNA. For example,less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less of the sample.Further, the approach should preferably be developed in a multiplexassay format in which one or more (preferably all) of the followingassays are included:

-   -   Assays for the detection of total amount of genomic equivalents        present in the sample, i.e., assays recognizing both maternal        and fetal DNA species;    -   Assays for the detection of fetal DNA isolated from a male        pregnancy, i.e., sequences specific for chromosome Y;    -   Assays specific for regions identified as differentially        methylated between the fetus and mother; or    -   Assays specific for regions known to be hypomethylated in all        tissues to be investigated, which can serve as a control for        restriction efficiency.

Other features of the assay may include one or more of the following:

-   -   For each assay, a target-specific, competitor oligonucleotide        that is identical, or substantially identical, to the target        sequence apart from a distinguishable feature of the competitor,        such as a difference in one or more nucleotides relative to the        target sequence. This oligonucleotide when added into the PCR        reaction will be co-amplified with the target and a ratio        obtained between these two PCR amplicons will indicate the        number of target specific DNA sequences (e.g., fetal DNA from a        specific locus) present in the maternal sample.    -   The amplicon lengths should preferably be of similar length in        order not to skew the amplification towards the shorter        fragments. However, as long as the amplification efficiency is        about equal, different lengths may be used.    -   Differentially methylated targets can be selected from Table 1        or from any other targets known to be differentially methylated        between mother and fetus. These targets can be hypomethylated in        DNA isolated from non-pregnant women and hypermethylated in        samples obtained from fetal samples. These assays will serve as        controls for the restriction efficiency.    -   The results obtained from the different assays can be used to        quantify one or more of the following:        -   Total number of amplifiable genomes present in the sample            (total amount of genomic equivalents);        -   The fetal fraction of the amplifiable genomes (fetal            concentration or percentage); or        -   Differences in copy number between fetally-derived DNA            sequences (for example, between fetal chromosome 21 and a            reference chromosome such as chromosome 3).

Examples of Assays Used in the Test

Below is an outline of the reaction steps used to perform a method ofthe technology, for example, as provided in FIG. 10. This outline is notintended to limit the scope of the technology. Rather it provides oneembodiment of the technology using the Sequenom® MassARRAY® technology.

-   -   1) DNA isolation from plasma samples.    -   2) Digestion of the DNA targets using methylation sensitive        restriction enzymes (for example, HhaI and HpaII).        -   For each reaction the available DNA was mixed with water to            a final volume of 25 ul.        -   10 ul of a reaction mix consisting of 10 units HhaI, 10            units HpaII and a reaction buffer were added. The sample was            incubated at an optimal temperature for the restriction            enzymes. HhaI and HpaII digest non-methylated DNA (and will            not digest hemi- or completely methylated DNA). Following            digestion, the enzymes were denatured using a heating step.    -   3) Genomic Amplification—PCR was performed in a total volume of        50 ul by adding PCR reagents (Buffer, dNTPs, primers and        polymerase). Exemplary PCR and extend primers are provided        below. In addition, synthetic competitor oligonucleotide was        added at known concentrations.    -   4) Replicates (optional)—Following PCR the 50 ul reaction was        split into 5 ul parallel reactions (replicates) in order to        minimize variation introduced during the post PCR steps of the        test. Post PCR steps include SAP, primer extension (MassEXTEND®        technology), resin treatment, dispensing of spectrochip and        MassARRAY.    -   5) Quantification of the Amplifiable Genomes—Sequenom MassARRAY®        technology was used to determine the amount of amplification        product for each assay. Following PCR, a single base extension        assay was used to interrogate the amplified regions (including        the competitor oligonucleotides introduced in step 3). Specific        extend primers designed to hybridize directly adjacent to the        site of interest were introduced. See extend primers provided        below. These DNA oligonucleotides are referred to as iPLEX®        MassEXTEND® primers. In the extension reaction, the iPLEX        primers were hybridized to the complementary DNA templates and        extended with a DNA polymerase. Special termination mixtures        that contain different combinations of deoxy- and        dideoxynucleotide triphosphates along with enzyme and buffer,        directed limited extension of the iPLEX primers. Primer        extension occurs until a complementary dideoxynucleotide is        incorporated.        -   The extension reaction generated primer products of varying            length, each with a unique molecular weight. As a result,            the primer extension products can be simultaneously            separated and detected using Matrix Assisted Laser            Desorption/Ionization, Time-Of-Flight (MALDI-TOF) mass            spectrometry on the MassARRAY® Analyzer Compact. Following            this separation and detection, SEQUENOM's proprietary            software automatically analyzes the data.    -   6) Calculating the amount and concentration of fetal nucleic        acid—Methods for calculating the total amount of genomic        equivalents present in the sample, the amount (and        concentration) of fetal nucleic acid isolated from a male        pregnancy, and the amount (and concentration) of fetal nucleic        based on differentially methylated targets are provided below        and in FIGS. 18 and 19.

The above protocol can be used to perform one or more of the assaysdescribed below. In addition to the sequences provided immediatelybelow, a multiplex scheme that interrogates multiple is provided inTable X below.

1) Assay for the Quantification of the Total Number of AmplifiableGenomic Equivalents in the Sample.

Targets were selected in housekeeping genes not located on thechromosomes 13, 18, 21, X or Y. The targets should be in a single copygene and not contain any recognition sites for the methylation sensitiverestriction enzymes.

Underlined sequences are PCR primer sites, italic is the site for thesingle base extend primer and bold letter (C) is the nucleotide extendedon human DNA

ApoE Chromosome 19:45409835-45409922 DNA target sequence withinterrogated nucleotide C in bold. All of the chromosome positionsprovided in this section are from the February 2009 UCSC Genome Build.(SEQ ID NO: 90) GATTGACAGTTTCTCCTTCCCCAGACTGGCCAATCACAGGCAGGAAGATGAAGGTTCTGTGGGCTGCGT TGCTGGTCACATTCCTGGC ApoE Forward Primer: (SEQ ID NO: 91)5′-ACGTTGGATG-TTGACAGTTTCTCCTTCCCC (Primer contains a 5′10 bp MassTag separated by a dash) ApoE Reverse Primer: (SEQ ID NO: 92)5′-ACGTTGGATG-GAATGTGACCAGCAACGCAG (Primer contains a 5′10 bp MassTag separated by a dash) ApoE Extension Primer:(SEQ ID NO: 93) 5′-GCAGGAAGATGAAGGTT [C/T]Primer extends C on human DNA targets and T on synthetic DNA targetsApoE synthetic competitor oligonucleotide: (SEQ ID NO: 94)5′-GATTGACAGTTTCTCCTTCCCCAGACTGGCCAATCACAGGCAGGAAGATGAAGGTTTTGTGGGCTGCGTTGCTGGTCACATTCCTGGC(Bold T at position 57 is different from human DNA)

2) Assay for the Quantification of the Total Number of Chromosome YSequences in the Sample.

Targets specific for the Y-chromosome were selected, with no similar orparalog sequences elsewhere in the genome. The targets should preferablybe in a single copy gene and not contain any recognition sites for themethylation sensitive restriction enzyme(s).

Underlined sequences are PCR primer sites, and italic nucleotide(s) isthe site for the single-base extend primer and bold letter (C) is thenucleotide extended on human DNA.

SRY on chrY: 2655628-2655717 (reverse complement) (SEQ ID NO: 95)GAGTTTTGGATAGTAAAATAAGTTTCGAACTCTGGCACCTTTCAATTT TGTCGCACTCTCCTTGTTTTTGACAATGCAATCATATGCTTC SRY Forward Primer: (SEQ ID NO: 96)5′-ACG-TGGATAGTAAAATAAGTTTCGAACTCTG (Primer contains a 5′3 bp MassTag separated by a dash) SRY Reverse Primer: (SEQ ID NO: 97)5′-GAAGCATATGATTGCATTGTCAAAAAC SRY Extension Primer: (SEQ ID NO: 98)5′-aTTTCAATTTTGTCGCACT [C/T]Primer extends C on human DNA targets and T on synthetic DNA targets. 5′Lower case “a” is an non-complementary nucleotideSRY synthetic competitor oligonucleotide: (SEQ ID NO: 99)5′-GAGTTTTGGATAGTAAAATAAGTTTCGAACTCTGGCACCTTTCAATTTTGTCGCACTTTCCTTGTTTTTGACAATGCAATCATATGCTTC

3) Assay for the Quantification of Fetal Methylated DNA SequencesPresent in the Sample.

Targets were selected in regions known to be differentially methylatedbetween maternal and fetal DNA. Sequences were selected to containseveral restriction sites for methylation sensitive enzymes. For thisstudy the HhaI (GCGC) and HpaII (CCGG) enzymes were used.

Underlined sequences are PCR primer sites, italic is the site for thesingle base extend primer and bold letter (C) is the nucleotide extendedon human DNA, lower case letter are recognition sites for themethylation sensitive restriction enzymes.

TBX3 on chr12: 115124905-115125001 (SEQ ID NO: 100)GAACTCCTCTTTGTCTCTGCGTGCccggcgcgcCCCCCTCccggTGGG TGATAAACCCACTCTGgcgccggCCATgcgcTGGGTGATTAATTTGCGA TBX3 Forward Primer:(SEQ ID NO: 101) 5′- ACGTTGGATG-TCTTTGTCTCTGCGTGCCC(Primer contains a 5′ 10 bp MassTag separated by a dash)TBX3 Reverse Primer: (SEQ ID NO: 102)5′- ACGTTGGATG-TTAATCACCCAGCGCATGGC (Primer contains a 5′10 bp MassTag separated by a dash) TBX3 Extension Primer:(SEQ ID NO: 103) 5′- CCCCTCCCGGTGGGTGATAAA [C/T]Primer extends C on human DNA targets and T on synthetic DNA targets. 5′Lower case “a” is a non-complementary nucleotideTBX3 synthetic competitor oligonucleotide: (SEQ ID NO: 104)5′-GAACTCCTCTTTGTCTCTGCGTGCCCGGCGCGCCCCCCTCCCGGTGGGTGATAAATCCACTCTGGCGCCGGCCATGCGCTGGGTGATTAATTT GCGA

4) Control Assay for the Enzyme Restriction Efficiency.

Targets were selected in regions known not to be methylated in anytissue to be investigated. Sequences were selected to contain no morethan one site for each restriction enzyme to be used.

Underlined sequences are PCR primer sites, italic nucleotide(s)represent the site for the single-base extend primer and bold letter (G)is the reverse nucleotide extended on human DNA, lower case letter arerecognition sites for the methylation sensitive restriction enzymes.

CACNA1G chr17:48637892-48637977(reverse complement) (SEQ ID NO: 105)CCATTGGCCGTCCGCCGTGGCAGTGCGGGCGGGAgcgcAGG GAGAGAACCACAGCTGGAATCCGATTCCCACCCCAAAACCCAGGA Hhal Forward Primer:(SEQ ID NO: 106) 5′- ACGTTGGATG-CCATTGGCCGTCCGCCGTG(Primer contains a 5′ 10 bp MassTag separated by a dash)Hhal Reverse Primer: (SEQ ID NO: 107)5′- ACGTTGGATG-TCCTGGGTTTTGGGGTGGGAA (Primer contains a 5′10 bp MassTag separated by a dash) Hhal Extension Primer:(SEQ ID NO: 108) 5′- TTCCAGCTGTGGTTCTCTCHhal synthetic competitor oligonucleotide: (SEQ ID NO: 109)5′-CCATTGGCCGTCCGCCGTGGCAGTGCGGGCGGGAGCGCAGA GAGAGAACCACAGCTGGAATCCGATTCCCACCCCAAAACCCAGGA

Validation Experiments

The sensitivity and accuracy of the present technology was measuredusing both a model system and clinical samples. In the differentsamples, a multiplex assay was run that contains 2 assays for total copynumber quantification, 3 assays for methylation quantification, 1 assayspecific for chromosome Y and 1 digestion control assay. See Tables X1and X2. Another multiplex scheme with additional assays is provided inTables Y1 and Y2.

TABLE X1 PCR Primers and Extend PrimersTable X1 discloses SEQ ID NOS 110-130, respectively, in order of appearance.Gene ID * First Primer Second Primer Extend Primer SOX14 MACGTTGGATGACATGGTCGGCCCCACGGAAT ACGTTGGATGCTCCTTCCTAGTGTGAGAACCGCAGGTTCCGGGGCTTGGG Hhal_CTRL D ACGTTGGATGACCCATTGGCCGTCCGCCGTACGTTGGATGTTTTGGGGTGGGAATCGGATT CGCAGGGAGAGAACCACAG TBX3 MACGTTGGATGGAACTCCTCTTTGTCTCTGCG ACGTTGGATGTGGCATGGCCGGCGCCAGACCCCTCCCGGTGGGTGATAAA SRY Y ACGTTGGATGCGCAGCAACGGGACCGCTACAACGTTGGCATCTAGGTAGGTCTTTGTAGCCAA AAAGCTGTAGGACAATCGGGT ALB TACGTTGCGTAGCAACCTGTTACATATTAA ACGTTGGATCTGAGCAAAGGCAATCAACACCCCATTTTTCTACATCCTTTGTTT EDG6 M ACGTTGGATGCATAGAGGCCCATGATGGTGGACGTTGGATGACCTTCTGCCCCTCTACTCCAA agAAGATCACCAGGCAGAAGAGG RNaseP TACGTTGGATGGTGTGGTCAGCTCTTCCC ACGTTGGCCCACATGTAATGTGTTGAAAAACTTGGAGAACAAAGGACACCG TTCAT AGCA TTA

TABLE X2 Competitor Oligonucleotide SequenceTable X2 discloses SEQ ID NOS 131-137, respectively, in order of appearance.Gene ID * Competitor Oligonucleotide Sequence SOX14 MGGTCGGCCCCACGGAATCCCGGCTCTGTGTGCGCCCAGGTTCCGGGGCTTGGGTGTTGCCGGTTCTCACACTAGGAAGGAGHhal_CTRL DCCATTGGCCGTCCGCCGTGGCAGTGCGGGCGGGAGCGCAGAGAGAGAACCACAGCTGGAATCCGATTCCCACCCCAAAATBX3 MGAACTCCTCTTTGTCTCTGCGTGCCCGGCGCGCCCCCCTCCCGGTGGGTGATAAATCCACTCTGGCGCCGGCCATGCSRY YGCAGCAACGGGACCGCTACAGCCACTGGACAAAGCCGTAGGACAATCGGGTAACATTGGCTACAAAGACCTACCTAGATGCALB TGCGTAGCAACCTGTTACATATTAAAGTTTTATTATACTACATTTTTCTACATCCTTTGTTTCAGAGTGTTGATTGCCTTTGCTCAGTATCTTCAG EDG6 MCCTTCTGCCCCTCTACTCCAAGCGCTACACCCTCTTCTGCCTGGTGATCTTTGCCGGCGTCCTGGCCACCATCATGGGCCTCTATGRNaseP TGTGTGGTCAGCTCTTCCCTTCATCACATACTTGGAGAACAAAGGACACCGTTATCCATGCTTTTTCAACACATTACATGTGGG

TABLE Y1 PCR Primers and Extend PrimersTable Y1 discloses SEQ ID NOS 138-167, respectively, in order of appearance.Gene ID * First Primer Second Primer Extend Primer EDG6 MACGTTGGATGTTCTGCCCCTCTACTCCAAG ACGTTGGATGCATAGAGGCCCATGATGGTGTTCTGCCTGGTGATCTT RNAseP T ACGTTGGATGTCAGCTCTTCCCTTCATCACACGTTGGATGCCTACCTCCCACATGTAATGT AACAAAGGACACCGTTA ApoE TACGTTGGATGTTGACAGTTTCTCCTTCCCC ACGTTGGATGGAATGTGACCAGCAACGCAGGCAGGAAGATGAAGGTT SOX14 M ACGTTGGATGCGGTCGGCCCCACGGAATACGTTGGATGCTCCTTCCTAGTGTGAGAACCG aAGGTTCCGGGGCTTGGG SRY no2 YACGTGGATAGTAAAATAAGTTTCGAACTCTG GAAGCATATGATTGCATTGTCAAAAACaTTTCAATTTTGTCGCACT SRY no1 Y ACGTTGGATGCACAGCTCACCGCAGCAACGACGTTGGATGCTAGGTAGGTCTTTGTAGCCAA AGCTGTAGGACAATCGGGT TBX3 MACGTTGGATGTCTTTGTCTCTGCGTGCCC ACGTTGGATGTTAATCACCCAGCGCATGGCCCCTCCCGGTGGGTGATAAA CACNA1G D ACGTTGGATGGACTGAGCCCCAGAACTCGACGTTGGATGGTGGGTTTGTGCTTTCCACG AGGGCCGGGGTCTGCGCGTG dig CTRL 1 DAPK1 digD ACGTTGGATGAAGCCAAGTTTCCCTCCGC ACGTTGGATGCTTTTGCTTTCCCAGCCAGGGAGGCACTGCCCGGACAAACC CTRL 2 ALB T ACGTTAGCGTAGCAACCTGTTACATATTAAACGTTGGATGCTGAGCAAAGGCAATCAACA CATTTTTCTACATCCTTTGTTT

TABLE Y2 Competitor Oligonucleotide SequenceTable Y2 discloses SEQ ID NOS 168-177, respectively, in order of appearance.Gene ID * Competitor EDG6 MCCTTCTGCCCCTCTACTCCAAGCGCTACACCCTCTTCTGCCTGGTGATCTTTGCCGGCGTCCTGGCCACCATCATGGGCCTCTATGRNAseP TGTGTGGTCAGCTCTTCCCTTCATCACATACTTGGAGAACAAAGGACACCGTTATCCATGCTTTTTCAACACATTACATGTGGGAGGTAGG ApoE TGATTGACAGTTTCTCCTTCCCCAGACTGGCCAATCACAGGCAGGAAGATGAAGGTTTTGTGGGCTGCGTTGCTGGTCACATTCCTGGC SOX14 MAAAACCAGAGATTCGCGGTCGGCCCCACGGAATCCCGGCTCTGTGTGCGCCCAGGTTCCGGGGCTTGGGTGTTGCCGGTTCTCACACTAGGAAGGAGC SRY no2 YGAGTTTTGGATAGTAAAATAAGTTTCGAACTCTGGCACCTTTCAATTTTGTCGCACTTTCCTTGTTTTTGACAATGCAATCATATGCTTC SRY no1 YGCAGCCAGCTCACCGCAGCAACGGGACCGCTACAGCCACTGGACAAAGCTGTAGGACAATCGGGTGACATTGGCTACAAAGACCTACCTAGATGC TBX3 MGAACTCCTCTTTGTCTCTGCGTGCCCGGCGCGCCCCCCTCCCGGTGGGTGATAAATCCACTCTGGCGCCGGCCATGCGCTGGGTGATTAATTTGCGA CACNA1G DGTGGGTTTGTGCTTTCCACGCGTGCACACACACGCGCAGACCCCGGCCCTTGCCCCGCCTACCTCCCCGAGTTCTGGGGCTCAGTCdig CTRL 1 DAPK1 dig DGCGCCAGCTTTTGCTTTCCCAGCCAGGGCGCGGTGAGGTTTGTCCGGGCAGTGCCTCGAGCAACTGGGAAGGCCAAGGCGGAGGGCTRL 2 AAAC ALB TGCGTAGCAACCTGTTACATATTAAAGTTTTATTATACTACATTTTTCTACATCCTTTGTTTTAGGGTGTTGATTGCCTTTGCTCAGTATCTTCAGC T = Assay for Total Amount; M = Assay for Methylationquantification; Y = Y-Chromosome Specific Assay; D = Digestion control

Model System Using Genomic DNA

In order to determine the sensitivity and accuracy of the method whendetermining the total number of amplifiable genomic copies in a sample,a subset of different DNA samples isolated from the blood ofnon-pregnant women was tested. Each sample was diluted to containapproximately 2500, 1250, 625 or 313 copies per reaction. The totalnumber of amplifiable genomic copies was obtained by taking the meanDNA/competitor ratio obtained from the three total copy number assays.The results from the four different samples are shown in FIG. 12.

To optimize the reaction, a model system was developed to simulate DNAsamples isolated from plasma. These samples contained a constant numberof maternal non-methylated DNA and were spiked with different amounts ofmale placental methylated DNA. The samples were spiked with amountsranging from approximately 0 to 25% relative to the maternalnon-methylated DNA. The results are shown in FIGS. 13A and B. Thefraction of placental DNA was calculated using the ratios obtained fromthe methylation assays (FIG. 13A), the SRY markers (FIG. 13B) and thetotal copy number assays. The primer sequences for the methylationassays (TBX), Y-chromosome assays (SRY) and total copy number (APOE) areprovided above. The model system demonstrated that the methylation-basedmethod performed equal to the Y-chromosome method (SRY markers), thusvalidating the methylation-based method as a sex-independent fetalquantifier.

Plasma Samples

To investigate the sensitivity and accuracy of the methods in clinicalsamples, 33 plasma samples obtained from women pregnant with a malefetus were investigated using the multiplex scheme from Table X. Foreach reaction, a quarter of the DNA obtained from a 4 ml extraction wasused in order to meet the important requirement that only a portion ofthe total sample is used.

Total Copy Number Quantification

The results from the total copy number quantification can be seen inFIGS. 14A and B. In FIG. 14A, the copy number for each sample is shown.Two samples (nos. 25 and 26) have a significantly higher total copynumber than all the other samples. In general, a mean of approximately1300 amplifiable copies/ml plasma was obtained (range 766-2055). FIG.14B shows a box-and-whisker plot of the given values, summarizing theresults.

Correlation Between Results Obtained from the Methylation Markers andthe Y-Chromosome Marker

In FIGS. 15A and B, the numbers of fetal copies for each sample areplotted. As all samples were from male pregnancies. The copy numbersobtained can be calculated using either the methylation or theY-chromosome-specific markers. As can be seen in FIG. 15B, thebox-and-whisker plot of the given values indicated minimal differencebetween the two different measurements.

The results showing the correlation between results obtained from themethylation markers and the Y-chromosome marker (SRY) is shown in FIG.16. Again, the methylation-based method performed equal to theY-chromosome method (SRY markers), further validating themethylation-based method as a sex-independent andpolymorphism-independent fetal quantifier. The multiplexed assaysdisclosed in Table X were used to determine the amount fetal nucleic.

Finally, the digestion efficiency was determined by using the ratio ofdigestion for the control versus the competitor and comparing this valueto the mean total copy number assays. See FIG. 17. Apart from sample 26all reactions indicate the efficiency to be above 99%.

Data Analysis

Mass spectra analysis was done using Typer 4 (a Sequenom softwareproduct). The peak height (signal over noise) for each individual DNAanalyte and competitor assay was determined and exported for furtheranalysis.

The total number of molecules present for each amplicon was calculatedby dividing the DNA specific peak by the competitor specific peak togive a ratio. (The “DNA” Peak in FIGS. 18 and 19 can be thought of asthe analyte peak for a given assay). Since the number of competitormolecules added into the reaction is known, the total number of DNAmolecules can be determined by multiplying the ratio by the number ofadded competitor molecules.

The fetal DNA fraction (or concentration) in each sample was calculatedusing the Y-chromosome-specific markers for male pregnancies and themean of the methylated fraction for all pregnancies. In brief, forchromosome Y, the ratio was obtained by dividing the analyte (DNA) peakby the competitor peak and multiplying this ratio by the number ofcompetitor molecules added into the reaction. This value was divided bya similar ratio obtained from the total number of amplifiable genomeequivalents determination (using the Assay(s) for Total Amount). SeeFIG. 18. Since the total amount of nucleic acid present in a sample is asum of maternal and fetal nucleic acid, the fetal contribution can beconsidered to be a fraction of the larger, background maternalcontribution. Therefore, translating this into the equation shown inFIG. 18, the fetal fraction (k) of the total nucleic acid present in thesample is equal to the equation: k=2×R/(1-2R), where R is the ratiobetween the Y-chromosome amount and the total amount. Since theY-chromosome is haploid and Assays for the Total Amount are determinedusing diploid targets, this calculation is limited to a fetal fractionsmaller than 50% of the maternal fraction.

In FIG. 19, a similar calculation for the fetal concentration is shownby using the methylation specific markers (see Assays for MethylationQuantification). In contrast to Y-chromosome specific markers, thesemarkers are from diploid targets, therefore, the limitations stated forthe Y-Chromosome Specific Assay can be omitted. Thus, the fetal fraction(k) can be determined using the equation: k=R(1−R), where R is the ratiobetween the methylation assay and the total assay.

Simulation

A first simple power calculation was performed that assumes ameasurement system that uses 20 markers from chromosome 21, and 20markers from one or more other autosomes. Starting with 100 copies offetal DNA, a measurement standard deviation of 25 copies and theprobability for a type I error to be lower than 0.001, it was found thatthe methods of the technology will be able to differentiate a diploidfrom a triploid chromosome set in 99.5% of all cases. The practicalimplementation of such an approach could for example be achieved usingmass spectrometry, a system that uses a competitive PCR approach forabsolute copy number measurements. The method can run 20 assays in asingle reaction and has been shown to have a standard deviation inrepeated measurements of around 3 to 5%. This method was used incombination with known methods for differentiating methylated andnon-methylated nucleic acid, for example, using methyl-binding agents toseparate nucleic acid or using methylation-sensitive enzymes to digestmaternal nucleic acid. FIG. 8 shows the effectiveness of MBD-FC protein(a methyl-binding agent) for capturing and thereby separating methylatedDNA in the presence of an excess of unmethylated DNA (see FIG. 8).

A second statistical power analysis was performed to assess thepredictive power of an embodiment of the Methylation-Based FetalDiagnostic Method described herein. The simulation was designed todemonstrate the likelihood of differentiating a group of trisomicchromosome 21 specific markers from a group of reference markers (forexample, autosomes excluding chromosome 21). Many parameters influencethe ability to discriminate the two populations of markers reliably. Forthe present simulation, values were chosen for each parameter that havebeen shown to be the most likely to occur based on experimentation. Thefollowing parameters and respective values were used:

Copy Numbers

-   -   Maternal copy numbers=2000    -   Fetal copy numbers for chromosomes other than 21, X and Y=200    -   Fetal copy numbers for chromosome 21 in case of euploid        fetus=200    -   Fetal copy numbers for chromosome 21 in case of aneuploid T21        fetus=300

Percent fetal DNA (before methylation-based enrichment)=10% (see above)

Methylation Frequency

-   -   Average methylation percentage in a target region for maternal        DNA=10%    -   Average methylation percentage in a target region for fetal        DNA=80%

Average percentage of non-methylated and non-digested maternal DNA(i.e., a function of restriction efficiency (among other things)=5%

Number of assays targeting chromosome 21=10

Number of assays targeting chromosomes other than 21, X and Y=10

The results are displayed in FIG. 20. Shown is the relationship betweenthe coefficient of variation (CV) on the x-axis and the power todiscriminate the assay populations using a simple t-test (y-axis). Thedata indicates that in 99% of all cases, one can discriminate the twopopulation (euploid vs. aneuploid) on a significance level of 0.001provided a CV of 5% or less. Based on this simulation, the methodrepresents a powerful noninvasive diagnostic method for the prenataldetection of fetal aneuploidy that is sex-independent and will work inall ethnicities (i.e., no allelic bias).

TABLE 1 MEAN LOG RATIO MICRO- GENE NAME CHROM START END CpG ISLAND ARRAYchr13 group00016 chr13 19773745 19774050 chr13: 19773518-19774214 0.19chr13 group00005 chr13 19290394 19290768 :- −0.89 CRYL1 chr13 1988709019887336 chr13: 19887007-19887836 −0.63 IL17D chr13 20193675 20193897chr13: 20193611-20194438 −1.01 CENPJ chr13 24404023 24404359 :- 0.57ATP8A2 chr13 25484475 25484614 chr13: 25484287-25484761 0.81 GSH1 chr1327265542 27265834 chr13: 27264549-27266505 0.57 PDX1 chr13 2739378927393979 chr13: 27392001-27394099 0.55 PDX1 chr13 27400459 27401165chr13: 27400362-27400744; 0.73 chr13: 27401057-27401374 MAB21L1 chr1334947737 34948062 chr13: 34947570-34948159 0.66 RB1 chr13 4779098347791646 chr13: 47790636-47791858 0.18 PCDH17 chr13 57104856 57106841chr13: 57104527-57106931 0.46 KLHL1 chr13 69579933 69580146 chr13:69579733-69580220 0.79 POU4F1 chr13 78079515 78081073 chr13:78079328-78079615; 0.66 chr13: 78080860-78081881 GPC6 chr13 9267740292678666 chr13: 92677246-92678878 0.66 SOX21 chr13 94152286 94153047chr13: 94152190-94153185 0.94 ZIC2 chr13 99439660 99440858 chr13:99439335-99440189; 0.89 chr13: 99440775-99441095 IRS2 chr13 109232856109235065 chr13: 109232467-109238181 −0.17 chr13 group00350 chr13109716455 109716604 chr13: 109716325-109716726 −0.37 chr13 group00385chr13 111595578 111595955 chr13: 111595459-111596131 0.87 chr13group00390 chr13 111756337 111756593 chr13: 111755805-111756697 0.71chr13 group00391 chr13 111759856 111760045 chr13: 111757885-1117606660.86 chr13 group00395 chr13 111808255 111808962 chr13:111806599-111808492; 0.96 chr13: 111808866-111809114 chr13 group00399chr13 112033503 112033685 chr13: 112032967-112033734 0.38 MCF2L chr13112724910 112725742 chr13: 112724782-112725121; −0.47 chr13:112725628-112725837 F7 chr13 112799123 112799379 chr13:112798487-112799566 −0.05 PROZ chr13 112855566 112855745 chr13:112855289-112855866 0.29 chr18 group00039 chr18 6919797 6919981 chr18:6919450-6920088 −0.38 CIDEA chr18 12244327 12244696 chr18:12244147-12245089 0.23 chr18 group00091 chr18 12901467 12901643 chr18:12901024-12902704 0.16 chr18 group00094 chr18 13126819 13126986 chr18:13126596-13127564 0.41 C18orf1 chr18 13377536 13377654 chr18:13377385-13377686 −0.12 KLHL14 chr18 28603978 28605183 chr18:28603688-28606300 0.83 CD33L3 chr18 41671477 41673011 chr18:41671386-41673101 −0.34 ST8SIA3 chr18 53171265 53171309 chr18:53170705-53172603 1.02 ONECUT2 chr18 53254808 53259810 chr18:53254152-53259851 0.74 RAX chr18 55086286 55086436 chr18:55085813-55087807 0.88 chr18 group00277 chr18 57151972 57152311 chr18:57151663-57152672 0.58 TNFRSF11A chr18 58203013 58203282 chr18:58202849-58203367 −0.33 NETO1 chr18 68685099 68687060 chr18:68684945-68687851 0.65 chr18 group00304 chr18 70133945 70134397 chr18:70133732-70134724 0.12 TSHZ1 chr18 71128742 71128974 chr18:71128638-71129076 0.23 ZNF236 chr18 72664454 72664736 chr18:72662797-72664893 −0.62 MBP chr18 72953150 72953464 chr18:72953137-72953402 0.6 chr18 group00342 chr18 74170347 74170489 chr18:74170210-74170687 −0.2 NFATC1 chr18 75385424 75386008 chr18:75385279-75386532 0.23 CTDP1 chr18 75596358 75596579 chr18:75596009-75596899 0.07 chr18 group00430 chr18 75653272 75653621 :- 0.52KCNG2 chr18 75760343 75760820 chr18: 75759900-75760988 0.01 OLIG2 chr2133317673 33321183 chr21: 33316998-33322115 0.66 OLIG2 chr21 3332759333328334 chr21: 33327447-33328408 −0.75 RUNX1 chr21 35180938 35185436chr21: 35180822-35181342; −0.68 chr21: 35182320-35185557 SIM2 chr2136994965 36995298 chr21: 36990063-36995761 0.83 SIM2 chr21 3699902536999410 chr21: 36998632-36999555 0.87 DSCR6 chr21 37300407 37300512chr21: 37299807-37301307 0.22 DSCAM chr21 41135559 41135706 chr21:41135380-41135816 1.03 chr21 group00165 chr21 43643421 43643786 chr21:43643322-43643874 1.14 AIRE chr21 44529935 44530388 chr21:44529856-44530472 −0.55 SUMO3 chr21 45061293 45061853 chr21:45061154-45063386 −0.41 C21orf70 chr21 45202815 45202972 chr21:45202706-45203073 −0.46 C21orf123 chr21 45671984 45672098 chr21:45671933-45672201 −0.63 COL18A1 chr21 45754383 45754487 chr21:45753653-45754639 −0.18 PRMT2 chr21 46911967 46912385 chr21:46911628-46912534 1.08 SIX2 chr2 45081223 45082129 chr2:45081148-45082287 1.15 SIX2 chr2 45084851 45085711 chr2:45084715-45084986; 1.21 chr2: 45085285-45086054 SOX14 chr3 138971870138972322 chr3: 138971738-138972096; 1.35 chr3: 138972281-138973691 TLX3chr5 170674439 170676431 chr5: 170674208-170675356; 0.91 chr5:170675783-170676712 FOXP4 chr6 41623666 41624114 chr6: 41621630-416241671.1 FOXP4 chr6 41636384 41636779 chr6: 41636244-41636878 1.32 chr7group00267 chr7 12576755 12577246 chr7: 12576690-12577359 0.94 NPY chr724290224 24291508 chr7: 24290083-24291605 0.93 SHH chr7 155291537155292091 chr7: 155288453-155292175 0.98 OSR2 chr8 100029764 100030536chr8: 100029673-100030614 1.21 GLIS3 chr9 4288283 4289645 chr9:4287817-4290182 1.24 PRMT8 chr12 3472714 3473190 chr12: 3470227-34732690.86 TBX3 chr12 113609153 113609453 chr12: 113609112-113609535 1.45chr12 group00801 chr12 118516189 118517435 chr12: 118515877-1185175951.1 PAX9 chr14 36201402 36202386 chr14: 36200932-36202536 0.89 SIX1chr14 60178801 60179346 chr14: 60178707-60179539 0.95 ISL2 chr1574420013 74421546 chr15: 74419317-74422570 1.08 DLX4 chr17 4539722845397930 chr17: 45396281-45398063 1.25 CBX4 chr17 75428613 75431793chr17: 75427586-75433676 1 EDG6 chr19 3129836 3130874 chr19:3129741-3130986 1.35 PRRT3 chr3 9963364 9964023 chr3: 9962895-9964619−0.85 MGC29506 chr5 138757911 138758724 chr5: 138755609-138758810 −0.63TEAD3 chr6 35561812 35562252 chr6: 35561754-35562413 −1.17 chr12group00022 chr12 1642456 1642708 chr12: 1642195-1642774 −1.33 CENTG1chr12 56406249 56407788 chr12: 56406176-56407818 −1.07 CENTG1 chr1256416146 56418794 chr12: 56416095-56416628; −0.94 chr12:56418745-56419001 MEAN MEAN METHYLATION RELATIVE MATERNAL PLACENTADIFFERENCE METHYLATION METHYLATION METHYLATION PLACENTA − PLACENTA TOGENE NAME EPITYPER EPITYPER MATERNAL MATERNAL chr13 group00016 0.22 0.320.1 HYPERMETHYLATION chr13 group00005 0.94 0.35 −0.59 HYPOMETHYLATIONCRYL1 0.74 0.21 −0.53 HYPOMETHYLATION IL17D 0.53 0.13 −0.39HYPOMETHYLATION CENPJ 0.17 0.49 0.32 HYPERMETHYLATION ATP8A2 0.16 0.430.27 HYPERMETHYLATION GSH1 0.13 0.19 0.05 HYPERMETHYLATION PDX1 0.06 0.20.14 HYPERMETHYLATION PDX1 0.12 0.26 0.14 HYPERMETHYLATION MAB21L1 0.110.17 0.06 HYPERMETHYLATION RB1 0.45 0.48 0.03 HYPERMETHYLATION PCDH170.15 0.21 0.06 HYPERMETHYLATION KLHL1 0.09 0.28 0.2 HYPERMETHYLATIONPOU4F1 0.12 0.23 0.11 HYPERMETHYLATION GPC6 0.06 0.19 0.13HYPERMETHYLATION SOX21 0.16 0.4 0.25 HYPERMETHYLATION ZIC2 0.13 0.350.22 HYPERMETHYLATION IRS2 0.73 0.38 −0.35 HYPOMETHYLATION chr13group00350 0.77 0.41 −0.36 HYPOMETHYLATION chr13 group00385 0.06 0.20.14 HYPERMETHYLATION chr13 group00390 0.12 0.34 0.22 HYPERMETHYLATIONchr13 group00391 0.11 0.36 0.25 HYPERMETHYLATION chr13 group00395 0.130.35 0.22 HYPERMETHYLATION chr13 group00399 0.26 0.43 0.18HYPERMETHYLATION MCF2L 0.91 0.33 −0.58 HYPOMETHYLATION F7 0.97 0.55−0.41 HYPOMETHYLATION PROZ 0.15 0.3 0.16 HYPERMETHYLATION chr18group00039 0.88 0.39 −0.49 HYPOMETHYLATION CIDEA 0.14 0.23 0.1HYPERMETHYLATION chr18 group00091 0.15 0.43 0.29 HYPERMETHYLATION chr18group00094 0.07 0.34 0.27 HYPERMETHYLATION C18orf1 0.95 0.69 −0.26HYPOMETHYLATION KLHL14 0.07 0.19 0.12 HYPERMETHYLATION CD33L3 0.49 0.44−0.05 HYPOMETHYLATION ST8SIA3 0.09 0.25 0.16 HYPERMETHYLATION ONECUT20.09 0.23 0.14 HYPERMETHYLATION RAX 0.11 0.26 0.16 HYPERMETHYLATIONchr18 group00277 0.08 0.21 0.13 HYPERMETHYLATION TNFRSF11A 0.88 0.28−0.6 HYPOMETHYLATION NETO1 0.09 0.22 0.13 HYPERMETHYLATION chr18group00304 0.93 0.92 −0.01 NOT CONFIRMED TSHZ1 0.95 0.92 −0.03 NOTCONFIRMED ZNF236 0.17 0.1 −0.07 HYPOMETHYLATION MBP 0.44 0.72 0.28HYPERMETHYLATION chr18 group00342 0.78 0.48 −0.3 HYPOMETHYLATION NFATC10.14 0.84 0.7 HYPERMETHYLATION CTDP1 0.97 0.96 −0.01 NOT CONFIRMED chr18group00430 0.24 0.62 0.39 HYPERMETHYLATION KCNG2 0.84 0.75 −0.09 NOTCONFIRMED OLIG2 0.11 0.2 0.09 HYPERMETHYLATION OLIG2 0.77 0.28 −0.49HYPOMETHYLATION RUNX1 0.14 0.07 −0.07 HYPOMETHYLATION SIM2 0.08 0.260.18 HYPERMETHYLATION SIM2 0.06 0.24 0.18 HYPERMETHYLATION DSCR6 0.040.14 0.11 HYPERMETHYLATION DSCAM 0.06 0.29 0.23 HYPERMETHYLATION chr21group00165 0.16 0.81 0.65 HYPERMETHYLATION AIRE 0.62 0.27 −0.35HYPOMETHYLATION SUMO3 0.55 0.46 −0.09 HYPOMETHYLATION C21orf70 0.96 0.51−0.46 HYPOMETHYLATION C21orf123 0.92 0.43 −0.49 HYPOMETHYLATION COL18A10.97 0.72 −0.25 HYPOMETHYLATION PRMT2 0.04 0.25 0.21 HYPERMETHYLATIONSIX2 0.08 0.36 0.28 HYPERMETHYLATION SIX2 0.07 0.35 0.28HYPERMETHYLATION SOX14 0.08 0.33 0.25 HYPERMETHYLATION TLX3 0.11 0.350.24 HYPERMETHYLATION FOXP4 0.07 0.27 0.2 HYPERMETHYLATION FOXP4 0.040.33 0.29 HYPERMETHYLATION chr7 group00267 0.08 0.26 0.17HYPERMETHYLATION NPY 0.09 0.3 0.21 HYPERMETHYLATION SHH 0.19 0.52 0.33HYPERMETHYLATION OSR2 0.08 0.43 0.35 HYPERMETHYLATION GLIS3 0.06 0.240.18 HYPERMETHYLATION PRMT8 0.07 0.23 0.16 HYPERMETHYLATION TBX3 0.090.56 0.48 HYPERMETHYLATION chr12 group00801 0.06 0.25 0.19HYPERMETHYLATION PAX9 0.11 0.32 0.21 HYPERMETHYLATION SIX1 0.1 0.33 0.22HYPERMETHYLATION ISL2 0.08 0.27 0.19 HYPERMETHYLATION DLX4 0.1 0.32 0.22HYPERMETHYLATION CBX4 0.07 0.27 0.21 HYPERMETHYLATION EDG6 0.04 0.870.83 HYPERMETHYLATION PRRT3 0.9 0.09 −0.81 HYPOMETHYLATION MGC29506 0.930.17 −0.76 HYPOMETHYLATION TEAD3 0.92 0.13 −0.8 HYPOMETHYLATION chr12group00022 0.66 0.09 −0.57 HYPOMETHYLATION CENTG1 0.95 0.19 −0.77HYPOMETHYLATION CENTG1 0.85 0.16 −0.69 HYPOMETHYLATION Information basedon the March 2006 human reference sequence (NCBI Build 36.1), which wasproduced by the International Human Genome Sequencing Consortium.

TABLE 2 GENE NAME CHROM START END SNPs chr13 chr13 19773745 19774050rs7996310; rs12870878 group00016 chr13 chr13 19290394 19290768rs11304938 group00005 CENPJ chr13 24404023 24404359 rs7326661 ATP8A2chr13 25484475 25484614 rs61947088 PDX1 chr13 27400459 27401165rs58173592; rs55836809; rs61944011 RB1 chr13 47790983 47791646rs2804094; rs4151432; rs4151433; rs4151434; rs4151435 PCDH17 chr1357104856 57106841 rs35287822; rs34642962; rs41292834; rs45500496;rs45571031; rs41292836; rs28374395; rs41292838 KLHL1 chr13 6957993369580146 rs3751429 POU4F1 chr13 78079515 78081073 rs11620410;rs35794447; rs2765065 GPC6 chr13 92677402 92678666 rs35689696;rs11839555; rs55695812; rs35259892 SOX21 chr13 94152286 94153047rs41277652; rs41277654; rs35276096; rs5805873; rs35109406 ZIC2 chr1399439660 99440858 rs9585309; rs35501321; rs9585310; rs7991728; rs1368511IRS2 chr13 109232856 109235065 rs61747993; rs1805097; rs9583424;rs35927012; rs1056077; rs1056078; rs34889228; rs1056080; rs1056081;rs12853546; rs4773092; rs35223808; rs35894564; rs3742210; rs34412495;rs61962699; rs45545638; rs61743905 chr13 chr13 111808255 111808962rs930346 group00395 MCF2L chr13 112724910 112725742 rs35661110;rs2993304; rs1320519; rs7320418; rs58416100 F7 chr13 112799123 112799379rs2480951; rs2476320 CIDEA chr18 12244327 12244696 rs60132277 chr18chr18 12901467 12901643 rs34568924; rs8094284; rs8094285 group00091C18orf1 chr18 13377536 13377654 rs9957861 KLHL14 chr18 28603978 28605183rs61737323; rs61737324; rs12960414 CD33L3 chr18 41671477 41673011rs62095363; rs2919643 ONECUT2 chr18 53254808 53259810 rs35685953;rs61735644; rs8084084; rs35937482; rs35427632; rs7232930; rs3786486;rs34286480; rs3786485; rs28655657; rs4940717; rs4940719; rs3786484;rs34040569; rs35542747; rs33946478; rs35848049; rs7231349; rs7231354;rs34481218; rs12962172; rs3911641 RAX chr18 55086286 55086436rs58797899; rs45501496 chr18 chr18 57151972 57152311 rs17062547group00277 TNFRSF11A chr18 58203013 58203282 rs35114461 NETO1 chr1868685099 68687060 rs4433898; rs34497518; rs35135773; rs6566677;rs57425572; rs36026929; rs34666288; rs10627137; rs35943684; rs9964226;rs4892054; rs9964397; rs4606820; rs12966677; rs8095606 chr18 chr1870133945 70134397 rs8086706; rs8086587; rs8090367; rs999332; rs17806420;rs58811193 group00304 TSHZ1 chr18 71128742 71128974 rs61732783;rs3744910; rs1802180 chr18 chr18 74170347 74170489 rs7226678 group00342NFATC1 chr18 75385424 75386008 rs28446281; rs56384153; rs4531815;rs3894049 chr18 chr18 75653272 75653621 rs34967079; rs35465647group00430 KCNG2 chr18 75760343 75760820 rs3744887; rs3744886 OLIG2chr21 33317673 33321183 rs2236618; rs11908971; rs9975039; rs6517135;rs2009130; rs1005573; rs1122807; rs10653491; rs10653077; rs35086972;rs28588289; rs7509766; rs62216114; rs35561747; rs7509885; rs11547332OLIG2 chr21 33327593 33328334 rs7276788; rs7275842; rs7275962;rs7276232; rs16990069; rs13051692; rs56231743; rs35931056 RUNX1 chr2135180938 35185436 rs2843956; rs55941652; rs56020428; rs56251824;rs13051109; rs13051111; rs3833348; rs7510136; rs743289; rs5843690;rs33915227; rs11402829; rs2843723; rs8128138; rs8131386; rs2843957;rs57537540; rs13048584; rs7281361; rs2843965; rs2843958 SIM2 chr2136994965 36995298 rs2252821 SIM2 chr21 36999025 36999410 rs58347144;rs737380 DSCAM chr21 41135559 41135706 rs35298822 AIRE chr21 4452993544530388 rs35110251; rs751032; rs9978641 SUMO3 chr21 45061293 45061853rs9979741; rs235337; rs7282882 C21orf70 chr21 45202815 45202972rs61103857; rs9979028; rs881318; rs881317 COL18A1 chr21 4575438345754487 rs35102708; rs9980939 PRMT2 chr21 46911967 46912385 rs35481242;rs61743122; rs8131044; rs2839379 SIX2 chr2 45081223 45082129 rs62130902SIX2 chr2 45084851 45085711 rs35417092; rs57340219 SOX14 chr3 138971870138972322 rs57343003 TLX3 chr5 170674439 170676431 rs11134682;rs35704956; rs2964533; rs35601828 FOXP4 chr6 41623666 41624114rs12203107; rs1325690 FOXP4 chr6 41636384 41636779 rs56835416 chr7 chr712576755 12577246 rs56752985; rs17149965; rs6948573; rs2240572group00267 NPY chr7 24290224 24291508 rs2390965; rs2390966; rs2390967;rs2390968; rs3025123; rs16146; rs16145; rs16144; rs13235842; rs13235935;rs13235938; rs13235940; rs13235944; rs36083509; rs3025122; rs16143;rs16478; rs16142; rs16141; rs16140; rs16139; rs2229966; rs1042552;rs5571; rs5572 SHH chr7 155291537 155292091 rs9333622; rs1233554;rs9333620; rs1233555 GLIS3 chr9 4288283 4289645 rs56728573; rs12340657;rs12350099; rs35338539; rs10974444; rs7852293 PRMT8 chr12 34727143473190 rs12172776 TBX3 chr12 113609153 113609453 rs60114979 chr12 chr12118516189 118517435 rs966246; rs17407022; rs970095; rs2711748 group00801PAX9 chr14 36201402 36202386 rs17104893; rs12883298; rs17104895;rs35510737; rs12882923; rs12883049; rs28933970; rs28933972; rs28933971;rs28933373; rs61734510 SIX1 chr14 60178801 60179346 rs761555 ISL2 chr1574420013 74421546 rs34173230; rs11854453 DLX4 chr17 45397228 45397930rs62059964; rs57481357; rs56888011; rs17638215; rs59056690; rs34601685;rs17551082 CBX4 chr17 75428613 75431793 rs1285243; rs35035500;rs12949177; rs3764374; rs62075212; rs62075213; rs3764373; rs3764372;rs55973291 EDG6 chr19 3129836 3130874 rs34728133; rs34573539; rs3826936;rs34914134; rs61731111; rs34205484 MGC29506 chr5 138757911 138758724rs11748963; rs7447765; rs35262202 CENTG1 chr12 56406249 56407788rs61935742; rs12318065; rs238519; rs238520; rs238521; rs808930;rs2640595; rs2640596; rs2640597; rs2640598; rs34772922 CENTG1 chr1256416146 56418794 rs11830475; rs34482618; rs2650057; rs2518686;rs12829991

TABLE 3 RELATIVE METHYLATION GENE NAME PLACENTA TO MATERNAL PRC2 TARGETCRYL1 HYPOMETHYLATION TRUE IL17D HYPOMETHYLATION TRUE GSH1HYPERMETHYLATION TRUE MAB21L1 HYPERMETHYLATION TRUE PCDH17HYPERMETHYLATION TRUE KLHL1 HYPERMETHYLATION TRUE POU4F1HYPERMETHYLATION TRUE SOX21 HYPERMETHYLATION TRUE ZIC2 HYPERMETHYLATIONTRUE CIDEA HYPERMETHYLATION TRUE KLHL14 HYPERMETHYLATION TRUE ONECUT2HYPERMETHYLATION TRUE RAX HYPERMETHYLATION TRUE TNFRSF11AHYPOMETHYLATION TRUE OLIG2 HYPERMETHYLATION TRUE OLIG2 HYPOMETHYLATIONTRUE SIM2 HYPERMETHYLATION TRUE SIM2 HYPERMETHYLATION TRUE SIX2HYPERMETHYLATION TRUE SIX2 HYPERMETHYLATION TRUE SOX14 HYPERMETHYLATIONTRUE TLX3 HYPERMETHYLATION TRUE SHH HYPERMETHYLATION TRUE OSR2HYPERMETHYLATION TRUE TBX3 HYPERMETHYLATION TRUE PAX9 HYPERMETHYLATIONTRUE SIX1 HYPERMETHYLATION TRUE ISL2 HYPERMETHYLATION TRUE DLX4HYPERMETHYLATION TRUE CBX4 HYPERMETHYLATION TRUE CENTG1 HYPOMETHYLATIONTRUE CENTG1 HYPOMETHYLATION TRUE

TABLE 4 SEQ ID GENE NO NAME SEQUENCE 1 chrCAGCAGGCGCGCTCCCGGCGAATCTGCCTGAATCGCCGTGAATGCGGTGGGGTGCAGGGCAGGGGCTGGTTTTCTCAGCCGGTCTTGGCTTTTC13grTCTTTCTCTCCTGCTCCACCAGCAGCCCCTCCGCGGGTCCCATGGGCTCCGCGCTCAGAACAGCCCGGAACCAGGCGCCGCTCGCCGCTCGCTGoup-GGGGCCACCCGCCTCTCCCCGGAACAGCCTCCCGCGGGCCTCTTGGCCTCGCACTGGCGCCCTCACCCACACATCGTCCCTTTATCCGCTCAGA00 CGCTGCAAAGGGCCTTCTGTCTC 016 2 CENGCTTTGGATTTATCCTCATTGGCTAAATCCCTCCTGAAACATGAAACTGAAACAAAGCCCTGAACCCCCTCAGGCTGAAAAGACAAACCCCGCCPJTGAGGCCGGGTCCCGCTCCCCACCTGGAGGGACCCAATTCTGGGCGCCTTCTGGCGACGGTCCCTGCTAGGGACGCTGCGCTCTCCGAGTGCGAGTTTTCGCCAAACTGATAAAGCACGCAGAACCGCAATCCCCAAACTAACACTGAACCCGGACCCGCGATCCCCAAACTGACAAGGGACCCGGAACAGCGACCCCCAAACCGACACGGGACTCGGGAACCGCTATCTCCAAAGGGCAGC 3 ATP8TTTCCACAACAGGGAGCCAGCATTGAGGCGCCCAGATGGCATCTGCTGGAAATCACGGGCCGCTGGTGAAGCACCACGCCTTACCCGACGTGGGA2 GAGGTGATCCCCCACCTCATCCCACCCCCTTCTGTCTGTCTCCTT 4 GSH1GCTGGACAAGGAGCGCTCACTGTAGCTCTGCTGTGGATTGTGTTGGGGCGAAGAGATGGGTAAGAGGTCAAAGTCGTAGGATTCTGGCGACCGCCTACCAAGGGATTGGGTCCACAGCACAGAGGTCTGATCGCTTCCTTCTCTGCTCTGCCACCTCCAGACAGCAGCTCTAACCAGCTGCCCAGCAGCAAGAGGATGCGCACGGCTTTCACCAGCACGCAGCTGCTAGAGCTGGAGCGCGAGTTCGCTTCTAATATGTACCTGTCCCGCCTACGTCGCATCGAGATCGCGA 5 PDX1TGCCTGACACTGACCCCAGGCGCAGCCAGGAGGGGCTTTGTGCGGGAGAGGGAGGGGGACCCCAGCTTGCCTGGGGTCCACGGGACTCTCTTCTTCCTAGTTCACTTTCTTGCTAAGGCGAAGGTCCTGAGGCAGGACGAGGGCTGAACTGCGCTGCAATCGTCCCCACCTCCAGCGAAACCCAGTTGAC 6 PDX1TCGGCGGAGAGACCTCGAGGAGAGTATGGGGAAAGGAATGAATGCTGCGGAGCGCCCCTCTGGGCTCCACCCAAGCCTCGGAGGCGGGACGGTGGGCTCCGTCCCGACCCCTTAGGCAGCTGGACCGATACCTCCTGGATCAGACCCCACAGGAAGACTCGCGTGGGGCCCGATATGTGTACTTCAAACTCTGAGCGGCCACCCTCAGCCAACTGGCCAGTGGATGCGAATCGTGGGCCCTGAGGGGCGAGGGCGCTCGGAACTGCATGCCTGTGCACGGTGCCGGGCTCTCCAGAGTGAGGGGGCCGTAAGGAGATCTCCAAGGAAGCCGAAAAAAGCAGCCAGTTGGGCTTCGGGAAAGACTTTTCTGCAAAGGAAGTGATCTGGTCCCAGAACTCCAGGGTTGACCCCAGTACCTGACTTCTCCGGGAGCTGTCAGCTCTCCTCTGTTCTTCGGGCTTGGCGCGCTCCTTTCATAATGGACAGACACCAGTGGCCTTCAAAAGGTCTGGGGTGGGGGAACGGAGGAAGTGGCCTTGGGTGCAGAGGAAGAGCAGAGCTCCTGCCAAAGCTGAACGCAGTTAGCCCTACCCAAGTGCGCGCTGGCTCGGCATATGCGCTCCAGAGCCGGCAGGACAGCCCGGCCCTGCTCACCCCGAGGAGAAATCCAACAGCGCAGCCTCCTGCACCTCCTTGCCCCAGAGAC 7 MABAGATCCCGGTGCATTTAAAGGCCGGCGTGATCTGCACCACGTACCTATCTCGGATTCTCAGTTTCACTTCGCTGGTGTCTGCCACCATCTTTAC21L1CACATCCCGGTAGCTACATTTGTCTACCGCTTGAGCCACCAGCGTCTGAAACCTGGACCGGATTTTGCGCGCCGAGAGGTAGCCGGAGGCGGTAATGAATTCCACCCAGAGGGACATGCTCCTCTTGCGCCCGTCGCTCAACTTCAGCACCGCGCAGCCGGGCAGTGAGCCATCGTCCACGAAGTTGAACACCCCCATTTGGTTGAGATAAAGCACCACTTCAAATTCGGT 8 RB1ACTATGCCTTGAGGGTCAAAACGTCTGGATTTCCTGATCGATGCTGTCGTCGCTGTCCACGGAGCTACTGTCGCCGTCAGAGCGGGAAGGCACGTTCAGGGAGTAGAAGCGTGGGCTTGCAGAAAGGGACCTGTTGCTGCCTTACATGGGGGCCGGCAGGGTAGTCTTGGAAATGCCCAAGATTGCTTCCGCGCGCGTCAGTTCAGCGGACGTGTCTGCCTGGCACGAGGACCGTTCTACAAACTCGTTCCTGGAAGCCGGGCTCGCTGGAGGCGGAGCTTTGGTTTCCTTCGGGAGCTTGTGGGGAATGGTCAGCGTCTAGGCACCCCGGGCAAGGGTCTGTGGCCTTGGTGGCCACTGGCTTCCTCTAGCTGGGTGTTTTCCTGTGGGTCTCGCGCAAGGCACTTTTTTGTGGCGCTGCTTGTGCTGTGTGCGGGGTCAGGCGTCCTCTCTCCTCCCGGCGCTGGGCCCTCTGGGGCAGGTCCCCGTTGGCCTCCTTGCGTGTTTGCCGCAGCTAGTACACCTGGATGGCCTCCTCAGTGCCGTCGTTGCTGCTGGAGTCTGACGCCTCGGGCGCCTGCGCCGCACTTGTGACTTGCTTTCCCCTTCTCAGGGCGCCAGCGCTCCTCTTGACCCCGCTTTTATTCTGTGGTGCTTCTGAAG9 PCDHGCAAGTCGGGTAGCTACCGGGTGCTGGAGAACTCCGCACCGCACCTGCTGGACGTGGACGCAGACAGCGGGCTCCTCTACACCAAGCAGCGCATC17GACCGCGAGTCCCTGTGCCGCCACAATGCCAAGTGCCAGCTGTCCCTCGAGGTGTTCGCCAACGACAAGGAGATCTGCATGATCAAGGTAGAGATCCAGGACATCAACGACAACGCGCCCTCCTTCTCCTCGGACCAGATCGAAATGGACATCTCGGAGAACGCTGCTCCGGGCACCCGCTTCCCCCTCACCAGCGCACATGACCCCGACGCCGGCGAGAATGGGCTCCGCACCTACCTGCTCACGCGCGACGATCACGGCCTCTTTGGACTGGACGTTAAGTCCCGCGGCGACGGCACCAAGTTCCCAGAACTGGTCATCCAGAAGGCTCTGGACCGCGAGCAACAGAATCACCATACGCTCGTGCTGACTGCCCTGGACGGTGGCGAGCCTCCACGTTCCGCCACCGTACAGATCAACGTGAAGGTGATTGACTCCAACGACAACAGCCCGGTCTTCGAGGCGCCATCCTACTTGGTGGAACTGCCCGAGAACGCTCCGCTGGGTACAGTGGTCATCGATCTGAACGCCACCGACGCCGATGAAGGTCCCAATGGTGAAGTGCTCTACTCTTTCAGCAGCTACGTGCCTGACCGCGTGCGGGAGCTCTTCTCCATCGACCCCAAGACCGGCCTAATCCGTGTGAAGGGCAATCTGGACTATGAGGAAAACGGGATGCTGGAGATTGACGTGCAGGCCCGAGACCTGGGGCCTAACCCTATCCCAGCCCACTGCAAAGTCACGGTCAAGCTCATCGACCGCAACGACAATGCGCCGTCCATCGGTTTCGTCTCCGTGCGCCAGGGGGCGCTGAGCGAGGCCGCCCCTCCCGGCACCGTCATCGCCCTGGTGCGGGTCACTGACCGGGACTCTGGCAAGAACGGACAGCTGCAGTGTCGGGTCCTAGGCGGAGGAGGGACGGGCGGCGGCGGGGGCCTGGGCGGGCCCGGGGGTTCCGTCCCCTTCAAGCTTGAGGAGAACTACGACAACTTCTACACGGTGGTGACTGACCGCCCGCTGGACCGCGAGACACAAGACGAGTACAACGTGACCATCGTGGCGCGGGACGGGGGCTCTCCTCCCCTCAACTCCACCAAGTCGTTCGCGATCAAGATTCTAGACGAGAACGACAACCCGCCTCGGTTCACCAAAGGGCTCTACGTGCTTCAGGTGCACGAGAACAACATCCCGGGAGAGTACCTGGGCTCTGTGCTCGCCCAGGATCCCGACCTGGGCCAGAACGGCACCGTATCCTACTCTATCCTGCCCTCGCACATCGGCGACGTGTCTATCTACACCTATGTGTCTGTGAATCCCACGAACGGGGCCATCTACGCCCTGCGCTCCTTTAACTTCGAGCAGACCAAGGCTTTTGAGTTCAAGGTGCTTGCTAAGGACTCGGGGGCGCCCGCGCACTTGGAGAGCAACGCCACGGTGAGGGTGACAGTGCTAGACGTGAATGACAACGCGCCAGTGATCGTGCTCCCCACGCTGCAGAACGACACCGCGGAGCTGCAGGTGCCGCGCAACGCTGGCCTGGGCTATCTGGTGAGCACTGTGCGCGCCCTAGACAGCGACTTCGGCGAGAGCGGGCGTCTCACCTACGAGATCGTGGACGGCAACGACGACCACCTGTTTGAGATCGACCCGTCCAGCGGCGAGATCCGCACGCTGCACCCTTTCTGGGAGGACGTGACGCCCGTGGTGGAGCTGGTGGTGAAGGTGACCGACCACGGCAAGCCTACCCTGTCCGCAGTGGCCAAGCTCATCATCCGCTCGGTGAGCGGATCCCTTCCCGAGGGGGTACCACGGGTGAATGGCGAGCAGCACCACTGGGACATGTCGCTGCCGCTCATCGTGACTCTGAGCACTATCTCCATCATCCTCCTA10 KLHATGCGCCCTCTGCACCCCTAGAGCCAGAAGACGCTAGGTGGGCTGCGCGCTCTGCCAGGCGAAGGCTGGAGCGCAGACGGCAAAGCCGCGCGTTTL1CAGCCGTGGTCGGGTCCGCAGGACCTGGGCGTGGGGACACCACCAGGCAGGAGCAGAGGCAGGACTGGGACGCCAAAAGCTGAGAATCCTCGATGCCCGCGCGAGAGCCCCGTGTTAT 11 POUTTCTGGAAACCGGGCCCCACTTGCAGGCCCGGCCACCTTGGGTTCTGGTGGCCGAAGCCGGAGCTGTGTTTCTCGCAGACTCGGGGAGCTACATT4F1GTGCGTAGGCAATTGTTTAGTTTGAAAGGAGGCACATTTCACCACGCAGCCAGCGCCCTGCATGCAGGAGAAGCCCCCAGGGCCCAGGGTCGGCTGGCTTTAGAGGCCACTTAGGTTGTTTTAAGCACATGTGAAAGGGCAGACAGCAGGGGAGCAGGATATGGGTAAGATCTTCGGGTCTCAGAACAGGGGCTGCCCTTGGGCTGTCCCGGCGCCCTGGGCTCTGACACTGAAGGGTGGAATGGAGGAAGGAATGGAGAAAGGACGGTGGAACTTTCGCTTCCCCTCTGGGCCGCCTTCCCAGGGTCATGCCTGAGCTGCTTTGATCCCAGTGTCGCGCATCTTGGTCCGCTACCTCCCAGGCGATAGCTACTGGGCTCCTCGCTGGCCTCACTGGGGGCCATCCCGGGCAGTGGCCTGCCCTCCGAGGCCCGCGGGACCCAGCCCAGAGCTGAGGTTGGAGTTCTCCGGGCCACGTTCCGGGTCGCTTAGGCTCGGAGATTTCCCGGAGACCGTCGTCCTCCCTTTCTGCTTGGCACTGCGGAGCTCCCTCGGCCTCTCTCCTCCTCTGGTCCCTAAGGCCCGGAGTGGTTGGCGGTACTGGGGCCCGTCGTCATCTCTGCTTCTAAGGCATTCAGACTGGGCTCCAGCTGGGACCGGCAGAGGAGGTTCTCAAGGAAACTGGTGGGAAATATAGTTTTCTTTCGTCTGGTCGTTTAATTTAAATGCAACTTCCCTTGGGGACATTTTCCTGGACGTTAACCAGACCACCTTGAGATGTCGTTGATGACCTAGAGACCCAGATGATGCGTCCCAGGAAAGTTCACTGCTGACTATTGTCACTCTTGGCGTTATATCTATAGATATAGACCTATGTACATATCTCCACCCTGATCTCTCCGTGGACATGAAACCCACCTACCTTGTGAAAGCCCTACGGGTGACACATGACTACTACGTCTCTGTCCCAACAGGGGCTGGGCCTCCCCTGCCTAATAGTTGCCAGGAGTTTCGCAGCCCAAGTGAATAATGTCTTATGGCTGAACGTGGCCAAGGACTCCTGTGATTTAGGTCCCAGGAGGAGCAGAGACGTCCCCGCCCCGCCTGGGCCCTGCCGCATTCAAAGCTGGAAGAAGGCGCTGATCAGAGAAGGGGCTTCCAGGTCCTGGGTTAGAACAACAACAAACAAACGAAACTCCACAACAGACACGCCTGCCCATGACCCCACGCAAGGACATAGGAAGTTCTGTCGCCTTCCTGCTCCGCGGATAGCCGCCTGCCGTCTGCTGCCACCAGAACGCACGGACGCTCGGGGTGGAGGTAGTCAATGGGCAGCAGGGGACCCCCAGCCCCCACAAGCGCGGCTCCGAGGACCTGGAAGCGGGTGCCTGTCGCTCTCCGCAGGCTCCGCTCTGCCTCCAGGAGCAAGATCCCCAAAAGGGTCTGGAAGCTGTGGAGAAAAC 12 GPC6TTTTTTAAACACTTCTTTTCCTTCTCTTCCTCGTTTTGATTGCACCGTTTCCATCTGGGGGCTAGAGGAGCAAGGCAGCAGCCTTCCCAGCCAGCCCTTGTTGGCTTGCCATCGTCCATCTGGCTTATAAAAGTTTGCTGAGCGCAGTCCAGAGGGCTGCGCTGCTCGTCCCCTCGGCTGGCAGAAGGGGGTGACGCTGGGCAGCGGCGAGGAGCGCGCCGCTGCCTCTGGCGGGCTTTCGGCTTGAGGGGCAAGGTGAAGAGCGCACCGGCCGTGGGGTTTACCGAGCTGGATTTGTATGTTGCACCATGCCTTCTTGGATCGGGGCTGTGATTCTTCCCCTCTTGGGGCTGCTGCTCTCCCTCCCCGCCGGGGCGGATGTGAAGGCTCGGAGCTGCGGAGAGGTCCGCCAGGCGTACGGTGCCAAGGGATTCAGCCTGGCGGACATCCCCTACCAGGAGATCGCAGGTAAGCGCGGGCGCGCTGCAGGGGCAGGCTGCAGCCCTCGGCTGCCGCACGTCCCACTGGCCGCCCGGCGTCCCCTTCCTTCCCCCTGTTGCTGAGTTGGTGCTCACTTTCTGCCACCGCTATGGGACTCCGCGTCTCCGTGTTGGGCGGCGGATGCTCCTGCGGCTTCTTCGGCGGGGGAAGGTGTGCGTCTCCGCCGCCTCATTGTGTGCACACGCGGGAGCACCCTGGCTCCCGCCTCCCGCTGCTCTCGCGCCCTTCTACCCCTTAGTTGATGGCTCAGGCCCGGCTGGCCAGGGAGCCCGGGTCACTCCGGGGCGGCTGCAAGGCGCAGACGGAGAGCCGAGCCGGGCGCTCACTCCGCGTTCTGGTTCGGGCAAACTTGGAAGAACTGCGACCGCAGTTTGCCCAGCGCCACAGTCTGAGTGGCGCCTTCTCCACTCCCGCCCTTGCGCCGGCAGGGGCGGTGGAGAGACGCGGAGGGCTCCCCCAGCCCCTCTCTCCCCTATCCGTCCTTCGGGCGACAGAGCGCCCGGCGCTCGGGCCGGGGGCGGGCAAGGCTGGGAGGGACCCTCGCCGGGGACCTGGCCTCTGGACGCCGGCGTTTCAAGGCTGGTTTGGGGACTTCACGGGCTGCCTGTTTCAGATGTGGGGCGGGCTTTCCCGTTAGGGTTCCTCAGTGCTTCCCCAGTTGCTGTTGGCCACTCAGGGCCCGGGGACACCCTGCCACCCGGTCTGGAGCCGGCCTCGTCTGCCAGCGAACAGCCAACTTTAGCGGGTGGCTCAGCTGGGGATT 13 SOXCACTCAGTGTGTGCATATGAGAGCGGAGAGACAGCGACCTGGAGGCCATGGGTGGGGGCGGGTGGTGAAGCTGCCGAAGCCTACACATACACTTA21GCTTTGACACTTCTCGTAGGTTCCAAAGACGAAGACACGGTGGCTTCAGGGAGACAAGTCGCAAGGGCGACTTTTCCAAGCGGGAGATGGTGAAGTCTTTGGACGTGTAGTGGGTAGGTGATGATCCCCGCAGCCGCCTGTAGGCCCGCAGACTTCAGAAAACAAGGGCCTTCTGTGAGCGCTGTGTCCTCCCCGGAATCCGCGGCTTAACACATTCTTTCCAGCTGCGGGGCCAGGATCTCCACCCCGCGCATCCGTGGACACACTTAGGGTCGCCTTTGTTTTGCGCAGTGATTCAAGTTGGGTAACCCTTGCTCAACACTTGGGAAATGGGGAGAATCTCCCCCACCCGCAACCTCCCGCACCCCAGGTTCCCAAAATCTGAATCTGTATCCTAGAGTGGAGGCAGCGTCTAGAAAGCAAAGAAACGGTGTCCAAAGACCCCGGAGAGTTGAGTGAGCGCAGATCCGTGACGCCTGCGGTACGCTAGGGCATCCAGGCTAGGGTGTGTGTGTGCGGGTCGGGGGGCGCACAGAGACCGCGCTGGTTTAGGTGGACCCGCAGTCCCGCCCGCATCTGGAACGAGCTGCTTCGCAGTTCCGGCTCCCGGCGCCCCAGAGAAGTTCGGGGAGCGGTGAGCCTAGCCGCCGCGCGCTCATGTTTATT 14 ZIC2AGTCACTCCAGGATCAGAGGCCGCGTCGGTTCTGCTTGGGGCATGGGCAGAGGGAGGCTGCTGGGGCCAAGCCCCGGCTGGACGCGAGGGAAGAAACTCGTCCCAGGACCCGCACGCCCATACCTGGCTGTCCCAGAGCTCTTCCCTAGGCCGGCACCTTCGCTCTTCCTCTTCCCCACCCCCTAGCCCTTTTGTCTCTTTTTCAGACGGATGTTTTCAGTCTCAAGTGGTTTTATTTTCCGCACAAAACCCTGAGATCAAGGGCAGATCACAGACTGTACCGGAGGCTCGGGTTTCCCTGGACTCTGTGCTGTTCTGCGTCCCAGGGTTGGCTAGGAAGGAAGGCCTGGGCCGGCGAGGTGACGGGTCTCCCGCCCAGGTCGGCAGGACGGGGGGAGGTGTGTCCCGGTAGGTCCCTGGTGAGCTCACCCGTGGCATCGGGGACCCGCGGGAACCCACCGGGCGCCCACTAGAGACTCGGGTCCTACCCTCCCCCACACTACTCCACCGAAATGATCGGAAGGGCGCGCTAGGCCTGCTTCCAAGGGCTCAGTGATAAAGGCCTCAAAATCACACTCCATCAAGACTTGGTTGAAGCTTTGGGTAGGTTTGTTGTTGTTGTTGTTGTTGTTTGTTTGTTTGTTTTAGCAGACACGTCCTGGAAAGAGGTCCTCAGAACCCAAAGGTTCAATAATGATTTGTGGATGGATTGATTATAGTCTGATATCGCTCTGGTTCCACAGAAACCCGGAGCTCCTTGGCCCACTGTTACCCCAGCAGACCTAAATGGACGGTTTCTGTTTTTCACTGGCAGCTCAGAACTGGACCGGAAGAAGTTCCCCTCCACTTCCCCCCTCCCGACACCAGATCATTGCTGGGTTTTTATTTTCGGGGGAAAAACAACAACAACAACAACAAAAAAAACACTAGGTCCTTCCAGACTGGATCAGGTGATCGGGCAAAAACCCTCAGGCTAGTCCGGCTGGGTGCCCGAGCATGAAAAGGCCTCCGTGGCCGTTTGAACAGGGTGTTGCAAATGAGAACTTTTGTAAGCCATAACCAGGGCATCCTGAGGGTCTGAGTTCACGGTCAAGGCTGTGGGCTACTAGGTCCAGCGAGTCCAGGCCTCGCCCCGCCCCCGAGCTGCCACAGCCAAGATCTTCGGCAGGGAATTCGAGACCAGGGTCCTCCCACTCCT 15 chrTTTCGTGCCGCTGTTTTCAATGCGCTAACGAGGCACGTTATTCTTAGCCGCGTCCGGGAGGGGATCACATTCCTGCGCAGTTGCGCTGCTG13GCGGAAGTGACTTGTTTTCTAACGACCCTCGTGACAGCCAGAGAATGTCCGTTTCTCGGAGCGCAGCACAGCCTGTCCCATCGAGAAGCCTgroup-CGGGTGAGGGGCCCGGTGGGCGCCCGGAGGCCGCTGGAGGGCTGTGGGAGGGACGGTGGCTCCCCACTCCCGTGGCGAAGGGCAGGCAAAC00385CAGAAGCCTCTTTTGAGAGCCGTTTGGGATTGAGACGAGTAAGCCACAGCGAGTGGTTAGAAGTAGGTTAGGAAGAAGGGGAGGTAAGAAAGCCGAGTAGGGTT 16 chrGTTCGGTGGACAAGGGGGCAGCGCCCACAGCAAGCCGGAAAGAGGGAGGCGCGGGGCCGCGCTTGGGGCCTGCCGCTGCACGCCAGCCTGGGCAA13AGAGCTGCCACCTTCTGCGGGCGAAGCGGGTCGGGACGCAGGACGGCAGCGGGGCTGGAGGCAGCTACGTGGGTCCACACCCCCATGCCCTGCAAgroup-GGCTCCTTGGCCCTGCTTCTCCTCTGTCTCGGCGGGAGAGGAGCAGCCTCGGTTTTACAGAATTTC 0039017 chrTGTGCCATTTAGTGAGAGGTGTTTTGGGCAAAGAATCAATTTAACTGTGACTGACCGACGGGCTTGACTGTATTAATTCTGCTACCGAAAAAAAA13AAAAAAAAAAAAGCAATGAGCCGCAAGCCTTGGACTCGCAGAGCTGCCGGTGCCCGTCCGAGAGCCCCACCAGCGCGGCTCACGCCTCAGTCTCgroup- 00391 18 chrAGAGTCCCAGTTCTGCAGGCCGCTCCAGGGCTAGGGGTAGAGATGGTGGCAGGTGGTGCGTCAACTCTCTAGGGAAGAGGAACTTGCATTACAAA13GACTTGTCTTTCTGAGCTGAAGTCAAAACGGGGGCGTCAAGCGCGCTCCGTTTGGCGGCGGTGGAGGGGCCGCGCGCCCGCGCTGTCCCAGCCGGgroup-AGCTGCCCTGGCTGGTGATTGGAGGTTTAACGTCCGGAATTCAGGCGCTTCTGCAGCTCAGATTTGCCGGCCAAGGGGCCTCAGTTGCAACTTTT00395CAAAATGGTGTTTCTGGAAAATAACAAATTCAGACTCAACTGGTGACAGCTTTTGGCTATAGAGAATGAAACTGCTTCCCTTTGGCGGTGGAACTCTTAAACTTCGAAGAGTGAAAGAATACAATGAAATAAAATGCCATAAGATCACTGGATTTTTCAGAAAAAGGAAGACCCCAAATTACTCCCAAAATGAGGCTTTGTAAATTCTTGTTAAAAATCTTTAAATCTCGAATTTCCCCCTACAACATCTGATGAGTGCTTTAAGAGCAAACGAGCAAATCCCACCTCGAGAATCAACAAACCCAAGCTCTGGCCAAGGCTCTCCCCGCGTTTTCTTCTCGTGACCTGGGGAATGTCCCGCCCCATCGCTCACCTGGCTCTTGTCATCTCGCTCATCTTGAAGTGACCCGTGGACAATGCTG 19 chrAGCTGCCCTCTGTGGCCATGAGCGGGTGTCCAGCCCCTTCCAAGGCTGCACCGGGGAGACGCTGGTTTTCTGCTCGCTGTGACCGAACAAAGCCC13CTAAGAGTCAGTGCGCGGAACAGAAGAGCCGGACCCCGACGGGCCGAGTCCCAACGTGAGGCACCCGGCAGAGAAAACACGTTCACGgroup- 00399 20 PROZCCTCGGCAGCACCGGCATGGCTGGAGGCCAGTACGGCCAGGTGTGGCGGGAGGGAGCGCCGTCTGGCTTGGGTCGTCCATCCTGACAGGACGCTGCAAGGGCAGGAGCCCCGCGCCCCGTGTCCTGCGCCCCCGCTCGAGGACAAGCCCCAGCCGCCGGTCTCCGCTGGGTTCCGACAG21 CIDCTTTAAGAGGCTGTGCAGGCAGACAGACCTCCAGGCCCGCTAGGGGATCCGCGCCATGGAGGCCGCCCGGGACTATGCAGGAGCCCTCATCAGGCEAGAGTGCCCCGCGTCCCCCTGATTGCCGTGCGCTTCCAATCGCCTTGCGTTCGGTGGCCTCATATTCCCCTGTGCGCCTCTAGTACCGTACCCCGCTCCCTTCAGCCCCCTGCTCCCCGCATTCTCTTGCGCTCCGCGACCCCGCGCACACACCCATCCGCCCCACTGGTGCCCAAGCCGTCCAGCCGCGCCCGCGGGCAGAGCCCAATCCCGTCCCGCGCCTCCTCACCCTCTTGCAGCTGGGCACAGGTACCAGGTGTGGCTCTTGCGAGGTG22 chrAGACTTGCAGAACTCGGGCCCCCTGGAGGAGACCTAACCGCCACGGTCTTGGGGAGGTTCCGGAGGGCCTCGGTTGTCTGCACTCCCAACACCAA18GAAACCCCTGAGACGCGAAGCTGCCAGCGTGCTGCCCTCAGAGCAGGGCGACGCAAAGCCAGCGGACCCCGGGGTGGCGGGgroup- 00091 23 chrTGCTCGGCTGGGGGGCTCGCTCCGCACTTTCGGTGCCAGAAAATGCCCAGAGGAGCGGGGCGGCCCCAGAGCCTCCTTTCGGGGCGCGAGGCCCG18GCGCGTGTGTACGGAGTCCAGTCCCCCCAGGGAGTGGGGTGCCCGCACCTTCCCCTCCGCGCTCGGAGCCACgroup- 00094 24 KLHTCTTGCACACCTGCTTGTAGTTCTGCACCGAGATCTGGTCGTTGAGGAACTGCACGCAGAGCTTGGTGACCTGGGGGATGTGCAGGATCTTGCTGL14ACCGACAGCACCTCCTCCACCGTGTCCAGGGACAGGGTCACGTTGGCCGTGTAGAGGTACTCGAGCACCAGGCGCAGCCCGATGGACGAGCAGCCCTGCAGCACCAGGTTGTTGATGGCCCGGGGGCTGGTCAGCAGCTTGTCGTCGGGGGAGGAAGAAGGAGTCCCGGGCTCCTCCTGCGGCGGCGGCTGCTGCTGCTGTGACGGCTGCTGCTGCGGCGGCTGCTGCTGGTCCTTGGGGGCCCCCAGGCCGTCCTGGCCGCCGACCCCTCCCCCGAGAGGGGGGTGGCTGGAGAAGAGCGATCGGAAGTACTGCGAGCAGGAGGCCAGCACGGCCTTGTGGCAATGGAACTGCTGGCCCTGGGCCGTCAGGGTCACGTCGCAAAACAGCTGCTTCCTCCACAGCAGGTTGAGGCCGTGCAGCAGGTTGTCGCTGTGGCTGGGGTCGAAGGTGGAGGTCCTGTCCCCGGATCTGGACATGGCGAGCTGACTCGGTGCACCTGGCTTTAAACCCTCCTCCAACCTGGCAGACAGGGGTGGGGGATGGGAGGGAGGGGAGCAGGGTGGTGGAGCGGGTGGGGTGTGGTCGGGGTGGGGAAGGGTGTGGAGGGGAGGGGAGGGCGAAGAACAAGAATCAAGGCTCAGCTTGACTCCCTCCTGGCGCGCTCCGGACCCCGACCCTAGGAGGAAAGTCCGAAGACGCTGGATCCGTGAGCGCCACCAGAAGGGCCCTGTCTGGGGTCCCGGCGCCGGTTCTGCGCCCTGCGGCTCCTCTCGCCACCTCCCACACACTTCGTCCCTCACTTTCCTAAAACCAACCACCTCAGCTCGGCTGTTGGCAGCAACAGCAGTGGCAGCAGCGACGGCAAAGTGGCGGCTGAGGCCGAGGCACCTCGTGGGCTCGTGTCCATGCCGGGCCAGATGAAGGGAAAGGCCGGGAAGTGGGGAGCCGGGGGTGCCCTGAAAGCTCAGAGGCGACCGACGGCGAAGGTTCCAGGTCAACTTGTGCCCGAAGCTTTGCTTTTCGCAGTTGGCCCAGTTTGGGGGAGGGGGTAGGAACAGGGGCCCGACCAGCGTGCGGGGTGTGCGAATCTTAGCTCTCCAAAAGCTG 25 ST8CCTCTGTGTTAGTGCCCTCGGGAATTTGGTTGATGGGGTGTTTG SIA3 26 ONETGATGTCGCACCTGAACGGCCTGCACCACCCGGGCCACACTCAGTCTCACGGGCCGGTGCTGGCACCCAGTCGCGAGCGGCCACCCTCGTCCTCACUT2TCGGGCTCGCAGGTGGCCACGTCGGGCCAGCTGGAAGAAATCAACACCAAAGAGGTGGCCCAGCGCATCACAGCGGAGCTGAAGCGCTACAGTATCCCCCAGGCGATCTTTGCGCAGAGGGTGCTGTGCCGGTCTCAGGGGACTCTCTCCGACCTGCTCCGGAATCCAAAACCGTGGAGTAAACTCAAATCTGGCAGGGAGACCTTCCGCAGGATGTGGAAGTGGCTTCAGGAGCCCGAGTTCCAGCGCATGTCCGCCTTACGCCTGGCAGGTAAGGCCGGGGCTAGCCAGGGGCCAGGCTGCTGGGAAGAGGGCTCCGGGTCCGGTGCTTGTGGCCCAAGTCTGCGCGCCGAGTCACTTCTCTTGATTCTTTCCTTCTCTTTCCTATACACGTCCTCTTTCTTCTCGTTTTTATTTCTTCTTCCATTTTCTCTTTCTCTTCCGCTCTTCCCCTACTTTCCCTTCTCCCTTTTCTTTTTCTTTCTTACTCTCTCCTTGTCCCTGAGCTTTCATTGACCGACCCCCCCCCATTTCATTCGCCCTCCCCTCAATGTGCCAACCTTTGCCCTATTTCCGATCTTCCCAGGTACTGGGAGGCGGGATGGGGGTGTGCGTTTTCCTCTAGGAGCCCTGTCTTTCCAAGACCCACAGAAACCAGGACCTGCCCTTATTCAAAACCCCATGCACTTCAAGTCTCTTTTAGACAACACATTTCAATTTTCCGGGCTGACTAGTCTCCCTGTGCAGAGGCAGTTGAGAGGCTTTGCTCTGCAGAGGGAAAAGAGCTCTCTACTCTCCCACCCACCATATAGGCAAACTTATTTGGTCATTGGCTGAAGGCACAGCCTTGCCCCCGCGGGGAACCGGCGGCCAGGATACAACAGCGCTCCTGGAGCCCATCTCTGGCCTTGGCGTTGGCGCAGGGACTTTCTGACCGGGCTTGAGGGGCTCGGGCCAGCTCCAATGTCACTACCTACAGCGAGGGCAGGGTGTAAGGTTGAGAAGGTCACATTCACCGCTTTGGGAGGACGTGGGAGAAGAGACTGAGGTGGAAAGCGCTTTGCCTTGCTCACCGGCCGTCCTTGCCCCGGTCCCAGCGTTTGCTGGGATTTGCCAGGATTTGCCGGGGCTCCGGGAGACCCTGAGCACTCGCAGGAAGAGGTGCTGAGAAATTAAAAATTCAGGTTAGTTAATGCATCCCTGCCGCCGGCTGCAGGCTCCGCCTTTGCATTAAGCGGGCGCTGATTGTGCGCGCCTGGCGACCGCGGGGAGGACTGGCGGCCCGCGGGAGGGGACGGGTAGAGGCGCGGGTTACATTGTTCTGGAGCCGGCTCGGCTCTTTGTGCCTCCTCTAGCGGCCAAGCTGCGAGGTACAGCCCTCTATTGTTCTAGGAGCACAGAAACCTCCTGTGTGGGCGGCGGGTGCGCGAGCTAGAGGGAAAGATGCAGTAGTTACTGCGACTGGCACGCAGTTGCGCGCTTTTGTGCGCACGGACCCCGCGCGGTGTGCGTGGCGACTGCGCTGCCCCTAGGAGCAAGCCACGGGCCCAGAGGGGCAAAATGTCCAGGTCCCCCGCTGGGAAGGACACACTATACCCTATGGCAAGCCAGGGTGGGCGACTTCCCATGGATCGGGTGGAGGGGGGTATCTTTCAGGATCGGCGGGCGGTCTAGGGGAACAATTCGTGGTGGCGATGATTTGCATAGCGCGGGTCTTGGGATGCGCGCGGTTCCGAGCCAGCCTCGCACAGCTCGCTTCCGGAGCTGCGAGCTCAGGTTTCCACCCCCGATCCCCCGGGCTTTCCTCGCACCGCTGAGCCCAGCTTGTGGGGTGCACTCGACCAACGCCCGACAGGGCTGGGGAATGTGACAGGCAGCAGGTTCACCCGGGCTTGGGGAGGGGGAGTTTCCGCTTTGACAGCATTTTCCTTTGCCGTCTGCTGGTGGATTCCTATTCCCAGTCGGTAATCGCCCCGCAGTGTTGATCTAAGAAGGTAAAGAAAACTAGGTTTCCCTGCAAAGAGCCTCCCCCAAATCGGCGGACTCCGGATACTTTGAGTGGATTTAGAAATTTATGTAATCTTTCTCCTTTAGTTTATTTTTCATCCTCTCCTACAGTTTTCTCTGATTTGCTGTTGGTTCGGGGCAAGATAAAGCAGCCAGTAGAGAGCGATAATAATAGCGGCGGGAAATGAACTGGAGACTGGCTGACAGTTCTTAACATTTTGTCATAGATCCCCCCGAATGTCCCAGGCTGTCTCTGGTGGGTTTTAGTACCCGCCGGCTTCTTGGGCACCGGGGACCAGAAGGAACTTGGCAGCTGGTCTTAGGGGTACAGTTAAAGGCAGGATGACAGCTATTCTCCTGCTCATCTCAGAGCGCTGCCGCCCCCTCATGCCGGTCGCGCAAAGAACACAGCTTTTAAAAAACACGTGCCTTCTGCCCATATAGGTCTGAAAGTGATGAGGAAAGTAATGCTTCGCCTATTAGCGAGTTTCAGCTTTTAAAATGATCCCAAGCGTTGCTGAGATGAGAAAGCGTGGCATCCCGGGGGTCCTCAGCCCCACCCGCGCCCATGGTGCAAGTCTGCAGGGACAGGCCCGGGACAGCACTGCCCACGCTGCTAGATTTTCCGCAGAGGATCGCTGAAGCTGCCTTCGTGGGAGACAGAATGCCTCCTCCAGCGAGTGGAAAAGGCCTGCTGAGGACCCCGCTTTGCTCGAGCATTCAAATGTGTGTCTGTTTTATTACCCTGGGTTGAAAAGGGACAAGAGCTTTAGCCTTTTTATCTGGCCATTTTATCAGCAACTACAAGTGTGTTGAGTGGTTATTATTACATAGGAGGCTTTTCAGTTTGGGGTCAGTAGATCAGTCTCTTCAGACACTGATGCAGAAGCTGGGACTGGTAAGTAGGTATTATGTGCTCGGAGCGCTAGGGGACAGGAGCAAATGGAGAAGAAAAGCGGAGGCTTTCTCCGCCCGGAGTATCGATCGGAATCCCCGCCGGTACGCCGCAGAGGGCCCTCGCCGTTGGGCCCCGGGGGTTTAACAAGCCCAGCCGCTCCGCAGGCGGCTCGGCCGGACTCTCAGACCGGTGCCTGGAAGACACCGTCCCTGCCCCCCTCCCGCCAAACCTGCCTCTTCTCTTTCTCTCATAGGTTATAGGTTCCCTTTCTCTCTCATTTTGGCCCCGCCCCCGGGTCCTGCCAAACAGCCAAGCAGGCCGGGGTTTAGGGGGCTCAGAATGAAGAGGTCTGATTTGGCCAGCGCCGGCAAAGCTCACCCTTAGGCGAGGTCACAACAGAGGCAGGTCCTTCCTGCCCAGCCTGCCGGTGTAGTCACAGCCAAGGGTGGCACTTGAAAGGAAAAGGGAGAAAACTTCGGAGAAATTTAGATTGCCCCAACGTTAGATTTCAGAGAAATTGACTCCAAATGCACGGATTCGTTCGGAAAGGGCGGCTAAGTGGCAGGTGGTTGCAACCCCGCCCGGTCGGGCCTTCGCAGAGGTTCCCCAAGACCAGCCCTTGCAGGGCGGTTTTCAGCAACCTGACAAGAGGCGGCCAAGACAAATTTCTGCGGGTTCGAGCACACACTCTCGGGCGTTGGGCCCCAGAGACCTCTAAACCAAGCACAAACAAGAAGGGAGTGAGAGAACCCAGGCTAGAACTTGCACGGGCATCCCACTGAGGAAAAGCGAGGCCTCGGTGGCAGGCATGTTTTCTTCCGACGCCCGAAAATCGAGCCGAGCGCCCGACTACATTTACTGCAGAGGTTTCCGCCTCCAGTGAGCCCGGATCCCCCAGCGGCCTGCCCGGAGCTGGTCTCCAGTCCCCGCCGTAGTCCGACGCACGGCCCTCTCCTGGCAGCAAGCTCCCAGCGGCCAGTCTGAAGCCAATTCTGTTCAGGCGGCCGAGGGCCCTTAGCCAACCCACCATGATGTCGCCTGGGCCACCTGATGCCCGCAGCGGCGGGACACGGCCCGGGCAGTGCGCAGTGGCTCCTGCTAGGGGCACCGCGTGCGTGCTTGTCTCCCGCTGCGCCGGGGACGTCCTTGGGTGACACGGGCCGCTGGGCACCTCCCAAGCCGAGGAAACGGACCCCCTTCGCAGAGTCTCGCGCCCACCCCCCAACCTCCCACCTCGTTTCTCGCTGCTAGGGCTCCCGACTCAGCCCACCTCTCCTGGCGGTTTAGTTAGGGATCAGAGCTGGAGAGGCTGAACGCAACCCGTGCCAGTACGGAACAGACGATATGTTTGCCTGCTAGCTGCTTGGATGAATAATTGAAAAGTTCGCTGCAGTCTGTGCTTCGTCAAGTCCCGGGTGCCGGGAGAACACCTTCCCAACACGCATCAGGGTGGGCGGGAGCGGGCAGAGGAGGCGGGACCCGAGGGAGGAGAGTGAACCCGAGCAGGAGAAGCAGCCCAGGCAGCCAGGCGCCCTCGATGCGAGAGGCTGGGCATTTATTTTTATTCCAGGCTTTCCACTGTGTGGTTATGTCACTTTCTCAAACAAATGTGTATATGGAGGGAGATCGATGCTGATAATGTTTAGAAGATTAAAAGAGCATTAATGCTGGCAACAATAACGTAAACGTGTGGACCCAGATTTCATTGATCTGGAACTTGATCCGGCGCGTTTCCAGTAAGCCCGACGGCGCGCTCTTCCCAGCAGAGCGCTCACCAGCGCCACGGCCCCGCGGTTTTCCAGCGGTGCCGCTTCGCCAGCTCTGCGCGGGTTCTCCCGTCTGACCGCAGCTCCTCCCCCGCGAGGCCCCAGCCCGCCTTACTTCCCCGAGGTTTTCTCCTCCTCTCGCGGGGCTCTCTGCCCTCTGCACCCCCTCCCCCGACCTCTGCACCACCCGCCCCTGTGCGCACACACCGCTACTTGCGCTTCCGGCGATCCGCCTG 27 RAXAACCGGAGATCTGCTTGGTGAACTGAGAGGAGTCCTTAGGAGAGCGGGGACGCCAGGGGCCGGGGGACACTTCGCTCTCGCCCTAGGGAAGGTGGTCTTGACGCTTTCTATTGAAGTCAAACTTGAAAATATCAGCTGCCGCTGGACTAT 28 chrCGTGAGCAGAACGCCCGCCCTGGAGCAGTTAGGACCGAAGGTCTCCGGAGAGTCGCCGGCGGTGCCAGGTAACGCAGAGGGCTCGGGTCGGGCCC18CGCTTCTGGGGCTTGGGACTCCGGGCGCGCGGAGCCAGCCCTCTGGGGCGAAATCCCCGGGCGGCGTGCGCGGTCCCTCTCCGCGCTGTGCTCTCgroup-CCAGCAACTCCCTGCCACCTCGACGAGCCTACCGGCCGCTCCGAGTTCGACTTCCTCGGACTTAGTGGGAGAAGGGGTTGGAAATGGGCTGCCGG00277 GACTGGGGGAGCTGCTCTCTGGAAGCAGGGAAGCTGGGGCGCACCGGGGCAGGT 29 NETTAGAAGAGGAAGACTCCTCTGGCCCCACTAGGTATCATCCGCGCTCTCCCGCTTTCCACCTGCGCCCTCGCTTGGGCCAATCTCTGCCGCACGTG01TCCATCCCTGAACTGCACGCTATCCTCCACCCCCGGGGGGTTCCTGCGCACTGAAAGACCGTTCTCCGGCAGGTTTTGGGATCCGGCGACGGCTGACCGCGCGCCGCCCCCACGCCCGGTTCCACGATGCTGCAATACAGAAAGTTTACGTCGGCCCCGACCCGCGCGGGACTGCAGGGTCCGCCGGAGCGCGGCGCAGAGGCTTTTCCTGCGCGTTCGGCCCCGGGAAAGGGGCGGGAGGGCTGGCTCCGGGAGCGCACGGGCGCGGCGGGGAGGGTACTCACTGTGAAGCACGCTGCGCCCATGGATCATGTCTGTGCGTTACACCAGAGGCTCCGGGCTCCACTAATTCCATTTAGAGACGGGAAGACTTCCAGTGGCGGGGGGAGGACAGGGTCGAGAGGTGTTAAAGACGCAAAGCAAGAAGGAAATAAAGGGGGGCCGAGAGGGAGACCGAGAGGAAGGGGGAGCTCCGAGCCCACGCTGCAGCCAGATCCGGATGAGTCCGTCCTCCGCCCCGGGCGGGCTCTCGCTCTCGCTGGCCCTCAGCGCCGCGCAGCCAGCAGCATCCCCACCGTGACGCTCGCATCACACCCGGGCGCCGGCCGCCACCATCCGCGCCGCCGCCGTCAGGACCCTCCTCCCGGGCATCGTCGCCGCCGCGGGGTCGGGAGGACGCGGCGCGCGGGAGGCGGCGGTCGCAGGGCGAGCCCCGGGACGCCCCGAGCCGGGGCCGGGGCCGGGGAGAGGGCGCAGCGAGGTGGGGGCCAGTCCAGACCGACGGCAGCGACGGAGCGGGCGGCGGCGGCGGCGCCGGCGGCGGCGGGGTGGCTCAGTCCCCAGTCTCAGACGCGCCGCGCAGCAGGTCGGAGCAGCCTCCCCGGGAGGATGTCCAGCGGCAGCGCTCCTCGCTCCAGCCCTTGGGGATCTTCCGCTGAGGCATTGAAGGCAGGAAGAAGGGGTCCGTCATCGGCTCGCCGGGCTGCGCGCCACCTCTGCTATCTTGCGGAAAGAGGAGCGGGTGGGTGGGCGTCTGGGAGGCGGGCTGGAGGGCGGTGCAGGGGAGCGGGGCGGCCGGGGGGGGGGCCGGGGGGCGGGGAAGGGAGGGAGGAGAAAGGAGCCGGAAGAGGGCAGAGTTACCAAATGGGCTCCTTAGTCATGGCTTGGGGCTCCACGACCCTCCTGGAAGCCCGGAGCCTGGGTGGGATAGCGAGGCTGCGCGCGGCCGGCGCCCCGGGGCTGGTGCGCGGCAGAATGGGGCCGCGGCGGCGGCAGCAAGGACATCCCAGCCGCGCGGATCTGGGGGAGGGGCGGGGAGGGGGTGAGGACCCGGCTGGGATCCGCGGCTCGGCCCGCCAGGGCGCAGAGAGAGGATGCAGCCGCAAATCCCGAGCCGGATCCTCGTGCCGGACGGAAGGCGTGGAAGCGGGAGGGGCCTTCGTGTGAAAATCCCTTGTGGGGTTTGGTGTTTCACTTTTTAAAGGTTAGACCTTGCGGGCTCTCTGCCTCCCACCCCTTCTTTTCCATCCGCGTAAAGGAACTGGGCGCCCCCTCTCCCTCCCTCCCTGGGGCGCAGGTTTCGCCGCGGACTCCGCGCTCAGCTTGGGAGACACGGCAGGGGCGCGCCCCAGGGAAAGGCGGCCGTAAAAGTTTCGCGGTTGAGCACTGGGCCTGATGTCCAGTCCCCCCACCAAATTACTCCTGCAAAGACGCGGGCTTCTTGCAATTGAGCCCCCCACCTCGAGGTATTTAAAACCACCCCAAGGCACACACGGACCCCCGTTCCCCCGCGCCACTTCCTCCTACAGGCTCGCGCGGCGCGTTAAAGTCTGGGAGACACGAGTTGCGGGGAAACAGCACCGGAAG 30 MBPAAGAAACAGCTCATTTCGGAGCTGAGGACAAGGCGTGGGAAGAAGACGCGTTTGGTTTCACCCAGGCGGGTGGCGGCAAAGCTGTGGGATGCGCGCTGCACACTCCTTCCGTCATCCCGTTCCCACCTTCCACACACACCTGCGGGAGGTCGGACATGTCCTGATTGCGTGTTCATCACGATGGCAAACCGAACATGAGGAGAACGCCACTGACGCTGGGTGCGCCGGCTTTCCCAGCCCTCGTGCATAACGGGGAGGGAGATGCAGAAGTTTTTTCCAACATCGGTGCAAAGGGGAAGCTGAGGTTTTCCTAT 31 NFATTCTGTCAGCTGCTGCCATGGGGCAGCGGGAAGGCCCTGGAGGGTGCCTGGGCTGTGTCTGGTCCCGGCCACGCGTCCCTGCAGCGTCTGAGACCTC1TGTGGAACACACTTGACCCGGCGCTGGGACGGGGTCGGCCCACACGCACCGCCAGCCCGCAGGAGTGAGGTGCAGGCTGCCGCTGGCTCCTTAGGCCTCGACAGCTCTCTTGAGGTCGGCCCTCCTCCCCTCCCGAGAGCTCAGCAGCCGCAGACCCAGGCAGAGAGAGCAAAGGAGGCTGTGGTGGCCCCCGACGGGAACCTGGGTGGCCGGGGGACACACCGAGGAACTTTCCGCCCCCCGACGGGCTCTCCCACCGAGGCTCAGGTGCTCGTGGGCAGCAAGGGGAAGCCCCATGGCCATGCCGCTTCCCTTTCACCCTCAGCGACGCGCCCTCCTGTGCCCGCGGGGAACAAGACGGCTCTCGGCGGCCATGCAGGCGGCCTGTCCCACGAACACGATGGAGACCTCAGACGCCGTCCCCACCCTGTCACTGTCACCATCACCCATCCTGTCCCCTCACGCCTCCCCACATCCCATCATTACTAC 32 chr18GAAGTAGAATCACAGTAAATGAGGAGTTAGGGAATTTAGGGTAGAGATTAAAGTAATGAACAGAGGAGGAGGCCTGAGACAGCTGCAGAGAGACCgroup-CTGTGTTCCCTGTGAGGTGAAGCGTCTGCTGTCAAAGCCGGTTGGCGCTGAGAAGAGGTACCGGGGGCAGCACCCGCCTCCTGGGAGAGGGATGG00430GCCTGCGGGCACCTGGGGGAACCGCACGGACACAGACGACACTATAAACGCGGGCGAGACATCAGGGACCGGGAAACAGAAGGACGCGCGTTTCGAGCAGCTGCCCAGTGGGCCACAAGCCCCGCCACGCCACAGCCTCTTCCCCTCAGCACGCAGAGA 33 OLIGTACTCCGGCGACGGGAGGATGTTGAGGGAAGCCTGCCAGGTGAAGAAGGGGCCAGCAGCAGCACAGAGCTTCCGACTTTGCCTTCCAGGCTCTAG2ACTCGCGCCATGCCAAGACGGGCCCCTCGACTTTCACCCCTGACTCCCAACTCCAGCCACTGGACCGAGCGCGCAAAGAACCTGAGACCGCTTGCTCTCACCGCCGCAAGTCGGTCGCAGGACAGACACCAGTGGGCAGCAACAAAAAAAGAAACCGGGTTCCGGGACACGTGCCGGCGGCTGGACTAACCTCAGCGGCTGCAACCAAGGAGCGCGCACGTTGCGCCTGCTGGTGTTTATTAGCTACACTGGCAGGCGCACAACTCCGCGCCCCGACTGGTGGCCCCACAGCGCGCACCACACATGGCCTCGCTGCTGTTGGCGGGGTAGGCCCGAAGGAGGCATCTACAAATGCCCGAGCCCTTTCTGATCCCCACCCCCCCGCTCCCTGCGTCGTCCGAGTGACAGATTCTACTAATTGAACGGTTATGGGTCATCCTTGTAACCGTTGGACGACATAACACCACGCTTCAGTTCTTCATGTTTTAAATACATATTTAACGGATGGCTGCAGAGCCAGCTGGGAAACACGCGGATTGAAAAATAATGCTCCAGAAGGCACGAGACTGGGGCGAAGGCGAGAGCGGGCTGGGCTTCTAGCGGAGACCGCAGAGGGAGACATATCTCAGAACTAGGGGCAATAACGTGGGTTTCTCTTTGTATTTGTTTATTTTGTAACTTTGCTACTTGAAGACCAATTATTTACTATGCTAATTTGTTTGCTTGTTTTTAAAACCGTACTTGCACAGTAAAAGTTCCCCAACAACGGAAGTAACCCGACGTTCCTCACACTCCCTAGGAGACTGTGTGCGTGTGTGCCCGCGCGTGCGCTCACAGTGTCAAGTGCTAGCATCCGAGATCTGCAGAAACAAATGTCTGAATTCGAAATGTATGGGTGTGAGAAATTCAGCTCGGGGAAGAGATTAGGGACTGGGGGAGACAGGTGGCTGCCTGTACTATAAGGAACCGCCAACGCCAGCATCTGTAGTCCAAGCAGGGCTGCTCTGTAAAGGCTTAGCAATTTTTTCTGTAGGCTTGCTGCACACGGTCTCTGGCTTTTCCCATCTGTAAAATGGGTGAATGCATCCGTACCTCAGCTACCTCCGTGAGGTGCTTCTCCAGTTCGGGCTTAATTCCTCATCGTCAAGAGTTTTCAGGTTTCAGAGCCAGCCTGCAATCGGTAAAACATGTCCCAACGCGGTCGCGAGTGGTTCCATCTCGCTGTCTGGCCCACAGCGTGGAGAAGCCTTGCCCAGGCCTGAAACTTCTCTTTGCAGTTCCAGAAAGCAGGCGACTGGGACGGAAGGCTCTTTGCTAACCTTTTACAGCGGAGCCCTGCTTGGACTACAGATGCCAGCGTTGCCCCTGCCCCAAGGCGTGTGGTGATCACAAAGACGACACTGAAAATACTTACTATCATCCGGCTCCCCTGCTAATAAATGGAGGGGTGTTTAACTACAGGCACGACCCTGCCCTTGTGCTAGCGCGGTTACCGTGCGGAAATAACTCGTCCCTGTACCCACACCATCCTCAACCTAAAGGAGAGTTGTGAATTCTTTCAAAACACTCTTCTGGAGTCCGTCCCCTCCCTCCTTGCCCGCCCTCTACCCCTCAAGTCCCTGCCCCCAGCTGGGGGCGCTACCGGCTGCCGTCGGAGCTGCAGCCACGGCCATCTCCTAGACGCGCGAGTAGAGCACCAAGATAGTGGGGACTTTGTGCCTGGGCATCGTTTACATTTGGGGCGCCAAATGCCCACGTGTTGATGAAACCAGTGAGATGGGAACAGGCGGCGGGAAACCAGACAGAGGAAGAGCTAGGGAGGAGACCCCAGCCCCGGATCCTGGGTCGCCAGGGTTTTCCGCGCGCATCCCAAAAGGTGCGGCTGCGTGGGGCATCAGGTTAGTTTGTTAGACTCTGCAGAGTCTCCAAACCATCCCATCCCCCAACCTGACTCTGTGGTGGCCGTATTTTTTACAGAAATTTGACCACGTTCCCTTTCTCCCTTGGTCCCAAGCGCGCTCAGCCCTCCCTCCATCCCCCTTGAGCCGCCCTTCTCCTCCCCCTCGCCTCCTCGGGTCCCTCCTCCAGTCCCTCCCCAAGAATCTCCCGGCCACGGGCGCCCATTGGTTGTGCGCAGGGAGGAGGCGTGTGCCCGGCCTGGCGAGTTTCATTGAGCGGAATTAGCCCGGATGACATCAGCTTCCCAGCCCCCCGGCGGGCCCAGCTCATTGGCGAGGCAGCCCCTCCAGGACACGCACATTGTTCCCCGCCCCCGCCCCCGCCACCGCTGCCGCCGTCGCCGCTGCCACCGGGCTATAAAAACCGGCCGAGCCCCTAAAGGTGCGGATGCTTATTATAGATCGACGCGACACCAGCGCCCGGTGCCAGGTTCTCCCCTGAGGCTTTTCGGAGCGAGCTCCTCAAATCGCATCCAGAGTAAGTGTCCCCGCCCCACAGCAGCCGCAGCCTAGATCCCAGGGACAGACTCTCCTCAACTCGGCTGTGACCCAGAATGCTCCGATACAGGGGGTCTGGATCCCTACTCTGCGGGCCATTTCTCCAGAGCGACTTTGCTCTTCTGTCCTCCCCACACTCACCGCTGCATCTCCCTCACCAAAAGCGAGAAGTCGGAGCGACAACAGCTCTTTCTGCCCAAGCCCCAGTCAGCTGGTGAGCTCCCCGTGGTCTCCAGATGCAGCACATGGACTCTGGGCCCCGCGCCGGCTCTGGGTGCATGTGCGTGTGCGTGTGTTTGCTGCGTGGTGTCGATGGAGATAAGGTGGATCCGTTTGAGGAACCAAATCATTAGTTCTCTATCTAGATCTCCATTCTCCCCAAAGAAAGGCCCTCACTTCCCACTCGTTTATTCCAGCCCGGGGGCTCAGTTTTCCCACACCTAACTGAAAGCCCGAAGCCTCTAGAATGCCACCCGCACCCCGAGGGTCACCAACGCTCCCTGAAATAACCTGTTGCATGAGAGCAGAGGGGAGATAGAGAGAGCTTAATTATAGGTACCCGCGTGCAGCTAAAAGGAGGGCCAGAGATAGTAGCGAGGGGGACGAGGAGCCACGGGCCACCTGTGCCGGGACCCCGCGCTGTGGTACTGCGGTGCAGGCGGGAGCAGCTTTTCTGTCTCTCACTGACTCACTCTCTCTCTCTCTCCCTCTCTCTCTCTCTCATTCTCTCTCTTTTCTCCTCCTCTCCTGGAAGTTTTCGGGTCCGAGGGAAGGAGGACCCTGCGAAAGCTGCGACGACTATCTTCCCCTGGGGCCATGGACTCGGACGCCAGCCTGGTGTCCAGCCGCCCGTCGTCGCCAGAGCCCGATGACCTTTTTCTGCCGGCCCGGAGTAAGGGCAGCAGCGGCAGCGCCTTCACTGGGGGCACCGTGTCCTCGTCCACCCCGAGTGACTGCCC34 SIM2TTAATTCGAAAATGGCAGACAGAGCTGAGCGCTGCCGTTCTTTTCAGGATTGAAAATGTGCCAGTGGGCCAGGGGCGCTGGGACCCGCGGTGCGGAAGACTCGGAACAGGAAGAAATAGTGGCGCGCTGGGTGGGCTGCCCCGCCGCCCACGCCGGTTGCCGCTGGTGACAGTGGCTGCCCGGCCAGGCACCTCCGAGCAGCAGGTCTGAGCGTTTTTGGCGTCCCAAGCGTTCCGGGCCGCGTCTTCCAGAGCCTCTGCTCCCAGCGGGGTCGCTGCGGCCTGGCCCGAAGGATTTGACTCTTTGCTGGGAGGCGCGCTGCTCAGGGTTCTG 35 SIM2CCGGTCCCCAGTTTGGAAAAAGGCGCAAGAAGCGGGCTTTTCAGGGACCCCGGGGAGAACACGAGGGCTCCGACGCGGGAGAAGGATTGAAGCGTGCAGAGGCGCCCCAAATTGCGACAATTTACTGGGATCCTTTTGTGGGGAAAGGAGGCTTAGAGGCTCAAGCTATAGGCTGTCCTAGAGCAACTAGGCGAGAACCTGGCCCCAAACTCCCTCCTTACGCCCTGGCACAGGTTCCCGGCGACTGGTGTTCCCAAGGGAGCCCCCTGAGCCTACCGCCCTTGCAGGGGGTCGTGCTGCGGCTTCTGGGTCATAAACGCCGAGGTCGGGGGTGGCGGAGCTGTAGAGGCTGCCCGCGCAGAAAGCTCCAGGATCCCAATATGTG 36 DSCRGCGCAGGTCCCCCCAGTCCCCGAGGGAGTGCGCCCGACGGAAACGCCCCTAGCCCGCGGGCCTCGCTTTCCTCTCCCGGGTTCCTGGGTCACTTC6 CCGCTGTCTC 37 DSTTCCCTCGCGGCTTTGGAAAGGGGGTGCAAATGCACCCTTCTGCGGGCCCGCTACCCGCTGCAACACCTGTGTTTCCTTTCTGGGCACCTTCTAGCAM GTTTCTAGATATTGCTGTGAATACGGTCCTCCGCTGTACAGTTGAAAACAAA 38 chr21TGGGAATTTAGGTCGGGCACTGCCGATATGTCGCCTTCCACAAGGCGGGCCCGGGCCTCTGCTGACCGTGCACCGGTCCTGGGGCTGGGTAATTCgroup-TGCAGCAGCAGCGCAGCCCATGCCGGGGAATTTGCGGGCAGAGGAGACAGTGAGGCCCGCGTTCTGTGCGGGAACTCCCGAGCTCACAGAGCCCA00165AGACCACACGGCTGCATCTGCTTGGCTGACTGGGCCAGGCCCACGCGTAGTAACCCGGACGTCTCTCTCTCACAGTCCCCTTGCGTCTGGCCAGGGAGCTGCCAGGCTGCACCCCGCGGTGGGGATCGGGAGAGGGGCAGTGTCGCCCATCCCCGGAAGGCTGAGCCTGGTGCAG39 PRMT2CGGTTTTCTCCTGGAGGACTGTGTTCAGACAGATACTGGTTTCCTTATCCGCAGGTGTGCGCGGCGCTCGCAAGTGGTCAGCATAACGCCGGGCGAATTCGGAAAGCCCGTGCGTCCGTGGACGACCCACTTGGAAGGAGTTGGGAGAAGTCCTTGTTCCCACGCGCGGACGCTTCCCTCCGTGTGTCCTTCGAGCCACAAAAAGCCCAGACCCTAACCCGCTCCTTTCTCCCGCCGCGTCCATGCAGAACTCCGCCGTTCCTGGGAGGGGAAGCCCGCGAGGCGTCGGGAGAGGCACGTCCTCCGTGAGCAAAGAGCTCCTCCGAGCGCGCGGCGGGGACGCTGGGCCGACAGGGGACCGCGGGGGCAGGGCGGAGAGGACCCGCCCTCGAGTCGGCCCAGCCCTAACACTCAGGAC 40 SIX2AGGGAATCGGGCTGACCAGTCCTAAGGTCCCACGCTCCCCTGACCTCAGGGCCCAGAGCCTCGCATTACCCCGAGCAGTGCGTTGGTTACTCTCCCTGGAAAGCCGCCCCCGCCGGGGCAAGTGGGAGTTGCTGCACTGCGGTCTTTGGAGGCCTAGGTCGCCCAGAGTAGGCGGAGCCCTGTATCCCTCCTGGAGCCGGCCTGCGGTGAGGTCGGTACCCAGTACTTAGGGAGGGAGGACGCGCTTGGTGCTCAGGGTAGGCTGGGCCGCTGCTAGCTCTTGATTTAGTCTCATGTCCGCCTTTGTGCCGGCCTCTCCGATTTGTGGGTCCTTCCAAGAAAGAGTCCTCTAGGGCAGCTAGGGTCGTCTCTTGGGTCTGGCGAGGCGGCAGGCCTTCTTCGGACCTATCCCCAGAGGTGTAACGGAGACTTTCTCCACTGCAGGGCGGCCTGGGGCGGGCATCTGCCAGGCGAGGGAGCTGCCCTGCCGCCGAGATTGTGGGGAAACGGCGTGGAAGACACCCCATCGGAGGGCACCCAATCTGCCTCTGCACTCGATTCCATCCTGCAACCCAGGAGAAACCATTTCCGAGTTCCAGCCGCAGAGGCACCCGCGGAGTTGCCAAAAGAGACTCCCGCGAGGTCGCTCGGAACCTTGACCCTGACACCTGGACGCGAGGTCTTTCAGGACCAGTCTCGGCTCGGTAGCCTGGTCCCCGACCACCGCGACCAGGAGTTCCTTCTTCCCTTCCTGCTCACCAGCCGGCCGCCGGCAGCGGCTCCAGGAAGGAGCACCAACCCGCGCTGGGGGCGGAGGTTCAGGCGGCAGGAATGGAGAGGCTGATCCTCCTCTAGCCCCGGCGCATTCACTTAGGTGCGGGAGCCCTGAGGTTCAGCCTGACTTTC 41 SIX2CACTACGGATCTGCCTGGACTGGTTCAGATGCGTCGTTTAAAGGGGGGGGCTGGCACTCCAGAGAGGAGGGGGCGCTGCAGGTTAATTGATAGCCACGGAAGCACCTAGGCGCCCCATGCGCGGAGCCGGAGCCGCCAGCTCAGTCTGACCCCTGTCTTTTCTCTCCTCTTCCCTCTCCCACCCCTCACTCCGGGAAAGCGAGGGCCGAGGTAGGGGCAGATAGATCACCAGACAGGCGGAGAAGGACAGGAGTACAGATGGAGGGACCAGGACACAGAATGCAAAAGACTGGCAGGTGAGAAGAAGGGAGAAACAGAGGGAGAGAGAAAGGGAGAAACAGAGCAGAGGCGGCCGCCGGCCCGGCCGCCCTGAGTCCGATCTTCCCTCCTTCCTGACCCTTCAGTTTCACTGCAAATCCACAGAAGCAGGTTTGCGAGCTCGAATACCTTTGCTCCACTGCCACACGCAGCACCGGGACTGGGCGTCTGGAGCTTAAGTCTGGGGGTCTGAGCCTGGGACCGGCAAATCCGCGCAGCGCATCGCGCCCAGTCTCGGAGACTGCAACCACCGCCAAGGAGTACGCGCGGCAGGAAACTTCTGCGGCCCAATTTCTTCCCCAGCTTTGGCATCTCCGAAGGCACGTACCCGCCCTCGGCACAAGCTCTCTCGTCTTCCACTTCGACCTCGAGGTGGAGAAAGAGGCTGGCAAGGGCTGTGCGCGTCGCTGGTGTGGGGAGGGCAGCAGGCTGCCCCTCCCCGCTTCTGCAGCGAGTTTTCCCAGCCAGGAAAAGGGAGGGAGCTGTTTCAGGAATTTCAGTGCCTTCACCTAGCGACTGACACAAGTCGTGTGTATAGGAAG 42 SOXGGAGCCTGAAGTCAGAAAAGATGGGGCCTCGTTACTCACTTTCTAGCCCAGCCCCTGGCCCTGGGTCCCGCAGAGCCGTCATCGCAGGCTCCTGC14CCAGCCTCTGGGGTCGGGTGAGCAAGGTGTTCTCTTCGGAAGCGGGAAGGGCTGCGGGTCGGGGACGTCCCTTGGCTGCCACCCCTGATTCTGCATCCTTTTCGCTCGAATCCCTGCGCTAGGCATCCTCCCCGATCCCCCAAAAGCCCAAGCACTGGGTCTGGGTTGAGGAAGGGAACGGGTGCCCAGGCCGGACAGAGGCTGAAAGGAGGCCTCAAGGTTCCTCTTTGCTACAAAGTGGAGAAGTTGCTCTACTCTGGAGGGCAGTGGCCTTTTCCAAACTTTTCCACTTAGGTCCGTAAGAAAAGCAATTCATACACGATCAGCGCTTTCGGTGCGAGGATGGAAAGAAACTTC43 TLX3TTTTCCTGTTACAGAGCTGAGCCCACTCATGTGGTGCCAAGTAGCGACTATCTCTCGGCCACCTCCACCCAGAGCAATGTGGGCGCCCCCAGCGGGTGGGAGCGATTGCCGAGCGGCGCAAGGGCGTTTAACGCCTAACCCCCTCCTCCTGGGTTGCCAAGCCGCTAGGTCGCCGTTTCCAACGTGGCTGCGCGGGACTGAAGTCCGACGACTCCTCGTCCTCAGTAGGAGACACACCTCCCACTGCCCCCAGCCACGCGAGCTATGGGCAGAATCGGGGCAACGGTAATATCTGGATGGGGCAGGCTCCCCTGAGGCTGTGCTTAAGAAAAAAGGAATCTGGAGTAGCCTGAGGGGCCCCACGAGGGGGCCTCCTTTGCGATCGTCTCCCAGCCTTAGGCCAAGGCTACGGAGGCAGGCGGCCGAGTGTTGGCGCCCAGCCCGGCCGAGGACTGGATGGAGGACGAGAAGCAGCCTGCCTCTGGGCGACAGCTGCGGACGCAGCCTCGCCGCCTCGCCGCCTCAGCCTCGGTCCCAGCGTCTCTAAAGCCGCGCCCATTTTACAGATGCAGGGCAGGGAGACAAGAGGCATCTCCGGGGGCCGAGTAGAATGATGGCGCGGGTTCTCCCGGCGCCCTGATTTCGAGGCTGCGCCCGGGGCCCTACATGCAGGCGGGGAGGCCTGGGCCGAAGGCGTCTGCAAGGAGGGGCGAGTCTGCCCGGTCCGGGCAGGGAGTGAGGCCACAGTCAGTTCTCCCTAGGAGGCCGCGCAGCGGGTAGGGTATGGGACTGGGGGACGCAACGGGGACCTGGCCGAATCAGAGCCCTCAGCAGAGAACGCCGAAAACTCTGGGGCCGGCCGCTCGCTTCCCGCTAGTGGGAATGGTTTCCGGTCATCCGTTCCCAGTCCAGCCCCGGGTAGGGAGCTCTGATTTGCAATGCACAGCACTTGCGAGGTTCGAATGCCCCCGCAATTTGCAGATGGAAATACTAAGCCTAGGCCGGGCGTGGTGGCTCAAGCCTATCATCTCAGCCCTTTGGGAGGCCAAGCCGGGAGGATTGTTTGAGCCCAAGAATTCAAAACCAGCCTGAGCAACATAGCGACCCCGTCTCTACAAAATAAAATAAAATAAATTATCCGGGCGTGGTGGCACGCGCCTGTGGTTCCAGCTACTCCGGAGGCTGAGGTGGGAGGATCGCTTGAGTCCGGGAGGTCGAGGCTACAGTGAGCCGTGATCGCACCACTGCACTCCAGCCTGGGCGACAGAGTGAGACCTTGTCTCAAAAAAGGAAAAAAAGAAAAAGAAAGTAAGCTTCAAAGAAGCTCTGATAATAGTTCTGGGTCGTGCAGCGGTGGCGGCCCCGCGCTCTCGCCCCTAAAGCAAGCGCTCTTTGTACTGGGTGGAGGAGCTTTGAGTAGTGAGGGTGGAGATGCAGCTTCGGGGTGGCGCAGCCACCCTGACACTAGGCCCGGGGTCGCAGTGGGACAGAAGAGTCTGCCGCTCTGACTTGGGCTCTGAGTTCCAAGGGCGCCCGGCACTTCTAGCCTCCCAGGCTTGCGCGCTGGCGCCTTTGCCATCCGTGCCGAAGTGGGGAGACCTAGCCGCGACCACCACGAGCGCAGCGGTGACACCCAGAGGTCCCACCGGGCCCCTGGGCAGGGTAACCTTAGCCTGTCCGCTTCGGCAGCTTTGCGAAGAGTGGCGCGCAGCTAGGGCTGAGGCTCTTGCGGACCTGCGGTCGAAGCAGGCGGCTGAGCCAGTTCGATCGCCAAGGCCTGGGCTGCCGACAGTGGTGCGCGCTCTGTTCCGCCGCGGCCGGGCCAGGCGCTCTGGAATAGCGATGGGGGGACACGGCCTCCAACTTTCTGCAGAGACCATCGGGCAGCTCCGGGCCTAAGCAGCGACCTCACCGAAGGTTCCTGGGAACCTTTGCCAAAATCCCAGCCTCTGCCTCGGTCCAGCTAAACCGTGTGTAAACAAGTGCACCAAG44 FOXATAAAGGACCGGGTAATTTCGCGGAATGCGGATTTTGAGACAGGCCCAGACGGCGGCGGATTCCCTGTGTCCCCCAACTGGGGCGATCTCGTGAAP4CACACCTGCGTCCCACCCCGATCCTAGGTTGGGGGGAAAGGGTATGGGAACCCTGAGCCCAGAGCGCGCCCCGCTCTTTCCTTTGCTCCCCGGCTTCCCTGGCCAGCCCCCTCCCGGCTGGTTTCCTCGCTCACTCGGCGCCTGGCGTTTCGGGCGTCTGGAGATCACCGCGTGTCTGGCACCCCAACGTCTAGTCTCCCCGCAGGTTGACCGCGGCGCCTGGAGCCGGGAATAGGGGTGGGGAGTCCGGAGAACCAAACCCGAGCCTGAAGTTGCCATTCGGGTGACTCCCGAGAAAGCCCGGGAGCATTTTGGCCAATGCGGGTTTTTACCTGAACTTCAGCATCTTCACC 45FOXAATTGGAAAACCCTGGTATTGTGCCTGTTTGGGGGAAGAAAACGTCAATAAAAATTAATTGATGAGTTGGCAGGGCGGGCGGTGCGGGTTCGCGGP4CGAGGCGCAGGGTGTCATGGCAAATGTTACGGCTCAGATTAAGCGATTGTTAATTAAAAAGCGACGGTAATTAATACTCGCTACGCCATATGGGCCCGTGAAAAGGCACAAAAGGTTTCTCCGCATGTGGGGTTCCCCTTCTCTTTTCTCCTTCCACAAAAGCACCCCAGCCCGTGGGTCCCCCCTTTGGCCCCAAGGTAGGTGGAACTCGTCACTTCCGGCCAGGGAGGGGATGGGGCGGTCTCCGGCGAGTTCCAAGGGCGTCCCTCGTTGCGCACTCGCCCGCCCAGGTTCTTTGAA 46 chr7GGGAAGCGATCGTCTCCTCTGTCAACTCGCGCCTGGGCACTTAGCCCCTCCCGTTTCAGGGCGCCGCCTCCCCGGATGGCAAACACTATAAAGTGgroup-GCGGCGAATAAGGTTCCTCCTGCTGCTCTCGGTTTAGTCCAAGATCAGCGATATCACGCGTCCCCCGGAGCATCGCGTGCAGGAGCCATGGCGCG00267GGAGCTATACCACGAAGAGTTCGCCCGGGCGGGCAAGCAGGCGGGGCTGCAGGTCTGGAGGATTGAGAAGCTGGAGCTGGTGCCCGTGCCCCAGAGCGCTCACGGCGACTTCTACGTCGGGGATGCCTACCTGGTGCTGCACACGGCCAAGACGAGCCGAGGCTTCACCTACCACCTGCACTTCTGGCTCGGTAAGGGACGGCGGGCGGCGGGACCCCGACGCACCAAGGCCGGCGAGGGGAGGGCGTAGGGGTCTGAGATTTGCAGGCGTGGGAGTAAAGGGGAGCCGCAAACTGAGCTA 47 NPYCTCAGGGGCGGGAAGTGGCGGGTGGGAGTCACCCAAGCGTGACTGCCCGAGGCCCCTCCTGCCGCGGCGAGGAAGCTCCATAAAAGCCCTGTCGCGACCCGCTCTCTGCACCCCATCCGCTGGCTCTCACCCCTCGGAGACGCTCGCCCGACAGCATAGTACTTGCCGCCCAGCCACGCCCGCGCGCCAGCCACCGTGAGTGCTACGACCCGTCTGTCTAGGGGTGGGAGCGAACGGGGCGCCCGCGAACTTGCTAGAGACGCAGCCTCCCGCTCTGTGGAGCCCTGGGGCCCTGGGATGATCGCGCTCCACTCCCCAGCGGACTATGCCGGCTCCGCGCCCCGACGCGGACCAGCCCTCTTGGCGGCTAAATTCCACTTGTTCCTCTGCTCCCCTCTGATTGTCCACGGCCCTTCTCCCGGGCCCTTCCCGCTGGGCGGTTCTTCTGAGTTACCTTTTAGCAGATATGGAGGGAGAACCCGGGACCGCTATCCCAAGGCAGCTGGCGGTCTCCCTGCGGGTCGCCGCCTTGAGGCCCAGGAAGCGGTGCGCGGTAGGAAGGTTTCCCCGGCAGCGCCATCGAGTGAGGAATCCCTGGAGCTCTAGAGCCCCGCGCCCTGCCACCTCCCTGGATTCTTGGGCTCCAAATCTCTTTGGAGCAATTCTGGCCCAGGGAGCAATTCTCTTTCCCCTTCCCCACCGCAGTCGTCACCCCGAGGTGATCTCTGCTGTCAGCGTTGATCCCCTGAAGCTAGGCAGACCAGAAGTAACAGAGAAGAAACTTTTCTTCCCAGACAAGAGTTTGGGCAAGAAGGGAGAAAAGTGACCCAGCAGGAAGAACTTCCAATTCGGTTTTGAATGCTAAACTGGCGGGGCCCCCACCTTGCACTCTCGCCGCGCGCTTCTTGGTCCCTGAGACTTCGAACGAAGTTGCGCGAAGTTTTCAGGTGGAGCAGAGGGGCAGGTCCCGACCGGACGGCGCCCGGAGCCCGCAAGGTGGTGCTAGCCACTCCTGGGTTCTCTCTGCGGGACTGGGACGAGAGCGGATTGGGGGTCGCGTGTGGTAGCAGGAGGAGGAGCGCGGGGGGCAGAGGAGGGAGGTGCTGCGCGTGGGTGCTCTGAATCCCCAAGCCCGTCCGTTGAGCCTTCTGTGCCTGCAGATGCTAGGTAACAAGCGACTGGGGCTGTCCGGACTGACCCTCGCCCTGTCCCTGCTCGTGTGCCTGGGTGCGCTGGCCGAGGCGTACCCCTCCAAGCCGGACAACCCGGGCGAGGACGCACCAG 48 SHHTGGAGAACCTTGGGCTCTGTGGCCTCAAAGGTAGGGGTGATTTCGAGGGGCCGGCACCTCACAGGGCAGGTTCCACCGCGGAAACGCAGTCATCGCCCAGCGACCCTGCTCCTGGCCCTCAGCCTCCCCCCAGGTTTCTTTTTCTCTTGAATCAAGCCGAGGTGCGCCAATGGCCTTCCTTGGGTCGGATCCGGGGGGCCAGGGCCAGCTTACCTGCTTTCACCGAGCAGTGGATATGTGCCTTGGACTCGTAGTACACCCAGTCGAAGCCGGCCTCCACCGCCAGGCGGGCCAGCATGCCGTACTTGCTGCGGTCGCGGTCAGACGTGGTGATGTCCACTGCGCGGCCCTCGTAGTGCAGAGACTCCTCTGAGTGGTGGCCATCTTCGTCCCAGCCCTCGGTCACCCGCAGTTTCACTCCTGGCCACTGGTTCATCACCGAGATGGCCAAAGCGTTCAACTTGTCCTTACACCTCTGCGAAGACAAGGGGACCCCCACCGACGGACACGTTAGCCTGGGCAACCGCCACCCCTCCCGGCCCCTCCATCAGCCT49 OSR2TCTCACGACCCATCCGTTAACCCACCGTTCCCAGGAGCTCCGAGGCGCAGCGGCGACAGAGGTTCGCCCCGGCCTGCTAGCATTGGCATTGCGGTTGACTGAGCTTCGCCTAACAGGCTTGGGGAGGGTGGGCTGGGCTGGGCTGGGCTGGGCTGGGTGCTGCCCGGCTGTCCGCCTTTCGTTTTCCTGGGACCGAGGAGTCTTCCGCTCCGTATCTGCCTAGAGTCTGAATCCGACTTTCTTTCCTTTGGGCACGCGCTCGCCAGTGGAGCACTTCTTGTTCTGGCCCCGGGCTGATCTGCACGCGGACTTGAGCAGGTGCCAAGGTGCCACGCAGTCCCCTCACGGCTTTCGGGGGGTCTTGGAGTCGGGTGGGGAGGGAGACTTAGGTGTGGTAACCTGCGCAGGTGCCAAAGGGCAGAAGGAGCAGCCTTGGATTATAGTCACGGTCTCTCCCTCTCTTCCCTGCCATTTTTAGGGCTTTCTCTACGTGCTGTTGTCTCACTGGGTTTTTGTCGGAGCCCCACGCCCTCCGGCCTCTGATTCCTGGAAGAAAGGGTTGGTCCCCTCAGCACCCCCAGCATCCCGGAAAATGGGGAGCAAGGCTCTGCCAGCGCCCATCCCGCTCCACCCGTCGCTGCAGCTCACCAATTACTCCTTCCTGCAGGCCGTGAACACCTTCCCGGCCACGGTGGACCACCTGCAGGGCCTGTACGGTCTCAGCGCGGTACAGACCATGCACATGAACCACTGGACGCTGGGGTATCCCAAT 50 GLITGGTTTCCTTTCGCTTCTCGCCTCCCAAACACCTCCAGCAAGTCGGAGGGCGCGAACGCGGAGCCAGAAACCCTTCCCCAAAGTTTCTCCCGCCAS3GGTACCTAATTGAATCATCCATAGGATGACAAATCAGCCAGGGCCAAGATTTCCAGACACTTGAGTGACTTCCCGGTCCCCGAGGTGACTTGTCAGCTCCAGTGAGTAACTTGGAACTGTCGCTCGGGGCAAGGTGTGTGTCTAGGAGAGAGCCGGCGGCTCACTCACGCTTTCCAGAGAGCGACCCGGGCCGACTTCAAAATACACACAGGGTCATTTATAGGGACTGGAGCCGCGCGCAGGACAACGTCTCCGAGACTGAGACATTTTCCAAACAGTGCTGACATTTTGTCGGGCCCCATAAAAAATGTAAACGCGAGGTGACGAACCCGGCGGGGAGGGTTCGTGTCTGGCTGTGTCTGCGTCCTGGCGGCGTGGGAGGTTATAGTTCCAGACCTGGCGGCTGCGGATCGCCGGGCCGGTACCCGCGAGGAGTGTAGGTACCCTCAGCCCGACCACCTCCCGCAATCATGGGGACACCGGCTTGGATGAGACACAGGCGTGGAAAACAGCCTTCGTGAAACTCCACAAACACGTGGAACTTGAAAAGACAACTACAGCCCCGCGTGTGCGCGAGAGACCTCACGTCACCCCATCAGTTCCCACTTCGCCAAAGTTTCCCTTCAGTGGGGACTCCAGAGTGGTGCGCCCCATGCCCGTGCGTCCTGTAACGTGCCCTGATTGTGTACCCCTCTGCCCGCTCTACTTGAAATGAAAACACAAAAACTGTTCCGAATTAGCGCAACTTTAAAGCCCCGTTATCTGTCTTCTACACTGGGCGCTCTTAGGCCACTGACAGAAACATGGTTTGAACCCTAATTGTTGCTATCAGTCTCAGTCAGCGCAGGTCTCTCAGTGACCTGTGACGCCGGGAGTTGAGGTGCGCGTATCCTTAAACCCGCGCGAACGCCACCGGCTCAGCGTAGAAAACTATTTGTAATCCCTAGTTTGCGTCTCTGAGCTTTAACTCCCCCACACTCTCAAGCGCCCGGTTTCTCCTCGTCTCTCGCCTGCGAGCAAAGTTCCTATGGCATCCACTTACCAGGTAACCGGGATTTCCACAACAAAGCCCGGCGTGCGGGTCCCTTCCCCCGGCCGGCCAGCGCGAGTGACAGCGGGCGGCCGGCGCTGGCGAGGAGTAACTTGGGGCTCCAGCCCTTCAGAGCGCTCCGCGGGCTGTGCCTCCTTCGGAAATGAAAACCCCCATCCAAACGGGGGGACGGAGCGCGGAAACCCGGCCCAAGTGCCGTGTGTGCGCGCGCGTCTG 51 PRMT8GAAAGCCATCCTTACCATTCCCCTCACCCTCCGCCCTCTGATCGCCCACCCGCCGAAAGGGTTTCTAAAAATAGCCCAGGGCTTCAAGGCCGCGCTTCTGTGAAGTGTGGAGCGAGCGGGCACGTAGCGGTCTCTGCCAGGTGGCTGGAGCCCTGGAAGCGAGAAGGCGCTTCCTCCCTGCATTTCCACCTCACCCCACCCCCGGCTCATTTTTCTAAGAAAAAGTTTTTGCGGTTCCCTTTGCCTCCTACCCCCGCTGCCGCGCGGGGTCTGGGTGCAGACCCCTGCCAGGTTCCGCAGTGTGCAGCGGCGGCTGCTGCGCTCTCCCAGCCTCGGCGAGGGTTAAAGGCGTCCGGAGCAGGCAGAGCGCCGCGCGCCAGTCTATTTTTACTTGCTTCCCCCGCCGCTCCGCGCTCCCCCTTCTCAGCAGTTGCACATGCCAGCTCTGCTGAAGGCATCAATGAAAACAGCAGTAG 52 TBX3ATCGAAAATGTCGACATCTTGCTAATGGTCTGCAAACTTCCGCCAATTATGACTGACCTCCCAGACTCGGCCCCAGGAGGCTCGTATTAGGCAGGGAGGCCGCCGTAATTCTGGGATCAAAAGCGGGAAGGTGCGAACTCCTCTTTGTCTCTGCGTGCCCGGCGCGCCCCCCTCCCGGTGGGTGATAAACCCACTCTGGCGCCGGCCATGCGCTGGGTGATTAATTTGCGAACAAACAAAAGCGGCCTGGTGGCCACTGCATTCGGGTTAAACATTGGCCAGCGTGTTCCGAAGGCTTGT 53 chr12ATCAACATCGTGGCTTTGGTCTTTTCCATCATGGTGAGTGAATCACGGCCAGAGGCAGCCTGGGAGGAGAGACCCGGGCGGCTTTGAGCCCCTGCgr-AGGGGAGTCCGCGCGCTCTCTGCGGCTCCCTTCCTCACGGCCCGGCCCGCGCTAGGTGTTCTTTGTCCTCGCACCTCCTCCTCACCTTTCTCGGGoup-CTCTCAGAGCTCTCCCCGCAATCATCAGCACCTCCTCTGCACTCCTCGTGGTACTCAGAGCCCTGATCAAGCTTCCCCCAGGCTAGCTTTCCTCT00TCTTTCCAGCTCCCAGGGTGCGTTTCCTCTCCAACCCGGGGAAGTTCTTCCGTGGACTTTGCTGACTCCTCTGACCTTCCTAGGCACTTGCCCGG801GGCTTCTCAACCCTCTTTTCTAGAGCCCCAGTGCGCGCCACCCTAGCGAGCGCAGTAAGCTCATACCCCGAGCATGCAGGCTCTACGTTCCTTTCCCTGCCGCTCCGGGGGCTCCTGCTCTCCAGCGCCCAGGACTGTCTCTATCTCAGCCTGTGCTCCCTTCTCTCTTTGCTGCGCCCAAGGGCACCGCTTCCGCCACTCTCCGGGGGGTCCCCAGGCGATTCCTGATGCCCCCTCCTTGATCCCGTTTCCGCGCTTTGGCACGGCACGCTCTGTCCAGGCAACAGTTTCCTCTCGCTTCTTCCTACACCCAACTTCCTCTCCTTGCCTCCCTCCGGCGCCCCCTTTTTAACGCGCCCGAGGCTGGCTCACACCCACTACCTCTTTAGGCCTTTCTTAGGCTCCCCGTGTGCCCCCCTCACCAGCAAAGTGGGTGCGCCTCTCTTACTCTTTCTACCCAGCGCGTCGTAGTTCCTCCCCGTTTGCTGCGCACTGGCCCTAACCTCTCTTCTCTTGGTGTCCCCCAGAGCTCCCAGGCGCCCCTCCACCGCTCTGTCCTGCGCCCGGGGCTCTCCCGGGAATGAACTAGGGGATTCCACGCAACGTGCGGCTCCGCCCGCCCTCTGCGCTCAGACCTCCCGAGCTGCCCGCCTCTCTAGGAGTGGCCGCTGGGGCCTCTAGTCCGCCCTTCCGGAGCTCAGCTCCCTAGCCCTCTTCAACCCTGGTAGGAACACCCGAGCGAACCCCACCAGGAGGGCGACGAGCGCCTGCTAGGCCCTCGCCTTATTGACTGCAGCAGCTGGCCCGGGGGTGGCGGCGGGGTGAGGTTCGTACCGGCACTGTCCCGGGACAACCCTTGCAGTTGC 54 PAX9ACAAATAAAACACCCTCTAGCTTCCCCTAGACTTTGTTTAACTGGCCGGGTCTCCAGAAGGAACGCTGGGGATGGGATGGGTGGAGAGAGGGAGCGGCTCAAGGACTTTAGTGAGGAGCAGGCGAGAAGGAGCACGTTCAGGCGTCAAGACCGATTTCTCCCCCTGCTTCGGGAGACTTTTGAACGCTCGGAGAGGCCCGGCATCTCACCACTTTACTTGGCCGTAGGGGCCTCCGGCACGGCAGGAATGAGGGAGGGGGTCCGATTGGACAGTGACGGTTTGGGGCCGTTCGGCTATGTTCAGGGACCATATGGTTTGGGGACAGCCCCAGTAGTTAGTAGGGGACGGGTGCGTTCGCCCAGTCCCCGGATGCGTAGGGAGGCCCAGTGGCAGGCAGCTGTCCCAAGCAGCGGGTGCGCGTCCCTGCGCGCTGTGTGTTCATTTTGCAGAGCCAGCCTTCGGGGAGGTGAACCAGCTGGGAGGAGTGTTCGTGAACGGGAGGCCGCTGCCCAACGCCATCCGGCTTCGCATCGTGGAACTGGCCCAACTGGGCATCCGACCGTGTGACATCAGCCGCCAGCTACGGGTCTCGCACGGCTGCGTCAGCAAGATCCTGGCGCGATACAACGAGACGGGCTCGATCTTGCCAGGAGCCATCGGGGGCAGCAAGCCCCGGGTCACTACCCCCACCGTGGTGAAACACATCCGGACCTACAAGCAGAGAGACCCCGGCATCTTCGCCTGGGAGATCCGGGACCGCCTGCTGGCGGACGGCGTGTGCGACAAGTACAATGTGCCCTCCGTGAGCTCCATCAGCCGCATTCTGCGCAACAAGATCGGCAACTTGGCCCAGCAGGGTCATTACGACTCATACAAGCAGCACCAGCCGACGCCGCAGCCAGCGCTGCCCTACAACCACATCTACTCGTACCCCAGCCCTATCACGGCGGCGGCCGCCAAGGTGCCCACGCCACCCGGGGTGC 55 SIX1AGGAGGCGCAACGCGCTGCCAGGGCGGCTTTATCCTGCCGCCACAGGGCGGGGACCAGCCCGGCAGCCGGGTGTCCAGCGCCGCTCACGTGCCTCGCCTGGAGCTTAGCTCTCAGACTCCGAAGAGGGCGACTGAGACTTGGGCCTGGGAGTTGGCTTCGGGGTACCCAAGGCGACGACAGCTGAGTTGTACCACGAAGCTCAGGCCGAGGCCTCCTCCCTTGTCTGGCCTTCGAATCCATACTGGCAGCCTCTCCTCTCAGGCACTCCGCGGGCCGGGCCACTAGGCCCCCTGCTCCTGGAGCTGCGCTATGATCCGGGTCTTGAGATGCGCGCGATTCTCTCTGAACCGGTGGAGAGGAGGCTCTGCCCCGCGCGGAGCGAGGACAGCGGCGCCCGAGCTTCCCGCGCCTCTCCAGGGCCCAATGGCAAGAACAGCCTCCGAAGTGCGCGGATGACAGGAAAAGATCTTCAGTTCTTCTGCCGCTAGAGAAGTGCGGGATACAAGCCTCTATTGGATCCACAACCTGGAGTCCTGCCTTCGGA56 ISL2ATCTGCGTGCCCTTTTCTGGGCGAGCCCTGGGAGATCCAGGGAGAACTGGGCGCTCCAGATGGTGTATGTCTGTACCTTCACAGCAAGGCTTCCCTTGGATTTGAGGCTTCCTATTTTGTCTGGGATCGGGGTTTCTCCTTGTCCCAGTGGCAGCCCCGCGTTGCGGGTTCCGGGCGCTGCGCGGAGCCCAAGGCTGCATGGCAGTGTGCAGCGCCCGCCAGTCGGGCTGGTGGGTTGTGCACTCCGTCGGCAGCTGCAGAAAGGTGGGAGTGCAGGTCTTGCCTTTCCTCACCGGGCGGTTGGCTTCCAGCACCGAGGCTGACCTATCGTGGCAAGTTTGCGGCCCCCGCAGATCCCCAGTGGAGAAAGAGGGCTCTTCCGATGCGATCGAGTGTGCGCCTCCCCGCAAAGCAATGCAGACCCTAAATCACTCAAGGCCTGGAGCTCCAGTCTCAAAGGTGGCAGAAAAGGCCAGACCTAACTCGAGCACCTACTGCCTTCTGCTTGCCCCGCAGAGCCTTCAGGGACTGACTGGGACGCCCCTGGTGGCGGGCAGTCCCATCCGCCATGAGAACGCCGTGCAGGGCAGCGCAGTGGAGGTGCAGACGTACCAGCCGCCGTGGAAGGCGCTCAGCGAGTTTGCCCTCCAGAGCGACCTGGACCAACCCGCCTTCCAACAGCTGGTGAGGCCCTGCCCTACCCGCCCCGACCTCGGGACTCTGCGGGTTGGGGATTTAGCCACTTAGCCTGGCAGAGAGGGGAGGGGGTGGCCTTGGGCTGAGGGGCTGGGTACAGCCCTAGGCGGTGGGGGAGGGGGAACAGTGGCGGGCTCTGAAACCTCACCTCGGCCCATTACGCGCCCTAAACCAGGTCTCCCTGGATTAAAGTGCTCACAAGAGAGGTCGCAGGATTAACCAACCCGCTCCCCCGCCCTAATCCCCCCCTCGTGCGCCTGGGGACCTGGCCTCCTTCTCCGCAGGGCTTGCTCTCAGCTGGCGGCCGGTCCCCAAGGGACACTTTCCGACTCGGAGCACGCGGCCCTGGAGCACCAGCTCGCGTGCCTCTTCACCTGCCTCTTCCCGGTGTTTCCGCCGCCCCAGGTCTCCTTCTCCGAGTCCGGCTCCCTAGGCAACTCCTCCGGCAGCGACGTGACCTCCCTGTCCTCGCAGCTCCCGGACACCCCCAACAGTATGGTGCCGAGTCCCGTGGAGACGTGAGGGGGACCCCTCCCTGCCAGCCCGCGGACCTCGCATGCTCCCTGCATGAGACTCACCCATGCTCAGGCCATTCCAGTTCCGAAAGCTCTCTCGCCTTCGTAATTATTCTATTGTTATTTATGAGAGAGTACCGAGAGACACGGTCTGGACAGCCCAAGGCGCCAGGATGCAACCTGCTTTCACCAGACTGCAGACCCCTGCTCCGAGGACTCTTAGTTTTTCAAAACCAGAATCTGGGACTTACCAGGGTTAGCTCTGCCCTCTCCTCTCCTCTCTACGTGGCCGCCGCTCTGTCTCTCCACGCCCCACCTGTGT 57 DLX4AGGTCTCTTCAGACTGCCCATTCTCCGGGCCTCGCTGAATGCGGGGGCTCTATCCACAGCGCGCGGGGCCGAGCTCAGGCAGGCTGGGGCGAAGATCTGATTCTTTCCTTCCCGCCGCCAAACCGAATTAATCAGTTTCTTCAACCTGAGTTACTAAGAAAGAAAGGTCCTTCCAAATAAAACTGAAAATCACTGCGAATGACAATACTATACTACAAGTTCGTTTTGGGGCCGGTGGGTGGGATGGAGGAGAAAGGGCACGGATAATCCCGGAGGGCCGCGGAGTGAGGAGGACTATGGTCGCGGTGGAATCTCTGTTCCGCTGGCACATCCGCGCAGGTGCGGCTCTGAGTGCTGGCTCGGGGTTACAGACCTCGGCATCCGGCTGCAGGGGCAGACAGAGACCTCCTCTGCTAGGGCGTGCGGTAGGCATCGTATGGAGCCCAGAGACTGCCGAGAGCACTGCGCACTCACCAAGTGTTAGGGGTGCCCGTGATAGACCGCCAGGGAAGGGGCTGGTTCGGAGGGAATTCCCGCTACCGGGAAGGTCGGAACTCGGGGTGATCAAACAAGGAATGCATCTCACCTCCGTGGGTGCTTGTGCTGCGCAAGGAATTATTACCGGAGCGGTTGCGATGGCCTTTGCCCGGCGACCCAAGAAGAGTAAGCAAACTACCGTCCACCCAGCGGATCAGGTCCAAT 58 CBX4GATGTCCTGTTTCTAGCAGCCTCCAGAGCCAAGCTAGGCGAGAGGCGTAGGAGGCAGAGAGAGCGGGCGCGGGAGGCCAGGGTCCGCCTGGGGGCCTGAGGGGACTTCGTGGGGTCCCGGGAGTGGCCTAGAAACAGGGAGCTGGGAGGGCCGGGAAGAGCTTGAGGCTGAGCGGGGGACGAACGGGCAGCGCAAAGGGGAGATGAACGGAATGGCCGAGGAGCCACGCATTCGCCTTGTGTCCGCGGACCCTTGTTCCCGACAGGCGACCAAGCCAAGGCCCTCCGGACTGACGCGGCCTGAGCAGCAGCGAGTGTGAAGTTTGGCACCTCCGGCGGCGAGACGGCGCGTTCTGGCGCGCGGCTCCTGCGTCCGGCTGGTGGAGCTGCTGCGCCCTATGCGGCCTGCCGAGGGCGCCGCCGAGGGCCCGCGAGCTCCGTGGGGTCGGGGTGGGGGGACCCGGGAGCGGACAGCGCGGCCCGAGGGGCAGGGGCAGGGGCGCGCCTGGCCTGGGGTGTGTCTGGGCCCCGGCTCCGGGCTCTTGAAGGACCGCGAGCAGGAGGCTTGCGCAATCCCTTGGCTGAGCGTCCACGGAGAAAGAAAAAGAGCAAAAGCAGAGCGAGAGTGGAGCGAGGGATGGGGGCGGGCAAAGAGCCATCCGGGTCTCCACCACCGCCCTGACACGCGACCCGGCTGTCTGTTGGGGACCGCACGGGGGCTCGGGCGAGCAGGGGAGGGAGGAGCCTGCGCGGGGCTCGTGTTCGCCCAGGAATCCCGGAGAAGCTCGAAGACGGTCTGGTGTTGAACGCACACGTGGACTCCATTTCATTACCACCTTGCAGCTCTTGCGCCACGGAGGCTGCTGCTGCCCGGCGGCTGCTACCCACCGAGACCCACGTGGCCCCTCCCCAGGGGTGTAGGGGTGACGGTTGTCTTCTGGTGACAGCAGAGGTGTTGGGTTTGCGACTGATCTCTAACGAGCTTGAGGCGCAAACCTAGGATTCCCTGAGTGTTGGGGTGCGGCGGGGGGGCAAGCAAGGTGGGACGACGCCTGCCTGGTTTCCCTGACTAGTTGCGGGGGGTGGGGGCCGGCTCTCAGGGGCCACCAGAAGCTGGGTGGGTGTACAGGAAAATATTTTTCTCCTGCCGTGTTTGGCTTTTTCCTGGCATTTTTGCCCAGGGCGAAGAACTGTCGCGCGGGGCAGCTCCACCGCGGAGGGAGAGGGGTCGCGAGGCTGGCGCGGGAAGCGCTGTAGGTGGCAGTCATCCGTCCACGCCGCACAGGCCGTCTGCGCCGTCGGACCATCGGGAGGTCTGCAGCAACTTTGTCCCGGCCAGTCCCCTTGTCCGGGAAGGGGCTGAGCTTCCCGACACTCTACCCTCCCCCTCTTGAAAATCCCCTGGAAAATCTGTTTGCAATGGGTGTTTCCGCGGCGTCCAGGTCTGGGCTGCCGGGGGAGGCCGAGCGGCTGCTGCAGCCTCCCTGCTGCCAGGGGCGTCGGACTCCGCTTCGCTCACTACGCCCAGGCCCCTCAGGGGCCCACGCTCAGGACTTCGGGGCCACACAGCAGGACCCGGTGCCCCGACGACGAGTTTGCGCAGGACCCGGGCTGGGCCAGCCGCGGAGCTGGGGAGGAAGGGGCGGGGGTCGGTGCAGCGGATCTTTTCTGTTGCTGCCTGTGCGGCGGCAGGAAGCGTCTTGAGGCTCCCCAAGACTACCTGAGGGGCCGCCCAAGCACTTCAGAAGCCCAAGGAGCCCCCGGCCACCCCCGCTCCTGGCCTTTTTGCCAACGACTTTGAAAGTGAAATGCACAAGCACCAGCAATTGACTTCCCTTCCGTGGTTATTTATTTTGTCTTTGTGGATGGTGGGCAGATGGGGAGAGAGGCCCCTACCTAACCTCGGTGGCTGGTCCCTAGACCACCCCTGCCAGCCGGTGTGGGGAGGAGCTCAGGTCCGCGGGAGAGCGAATGGGCGCCAGGAGGTGGGACAGAATCCTGGGAAGGTACAGCGGACGCCCTGGAAGCTCCCCTGATGCCCCAGAGGGCCCTTCCTGGGAAACCTCCCGGGGGGGTGCCCCATACCATCCCACCCGGCTGTCTTGGCCCCTCCCAGGGAGCCGCAGGAGAAACTAGCCCTACACCTGGGATTCCCAGAGCCTTCTGCTGGGGCTCCTGCCCCCGACTTCGGATAACCAGCTCCGCACAGGTCCCCGAGAAGGGCCGCTGGCCTGCTTATTTGATACTGCCCCCTCCCAGACAGGGGCTGGTCGAGCCCCTGGTTCTGCTGCCAGACTGAAGCCTTCCAGACGCCACCTCGGTTTGGGCCCCCAGGGCCCTCAGGGGCCCCAGGAGAGGAGAGCTGCTATCTAGCTCAGCCACAGGCTCGCTCCTGGTGGGGGCCAGGCTGAAGGAGTGGACCCTGGAGAGGTCGGGAACCTTTTAACAGCCGTGGGCTGGAGGGTGGCTACTAAGTGTTCGGTCTGGGAAGAGGCATGACCCGCACCATCCCGGGGAAATAAACGACTTCTTAAGGGAATCTTCTCGCTGAGCGGGTGCTCTGGGCCAGGAGATTGCCACCGCCAGCCCACGGAACCCAGATTTGGGCTCTGCCTTGAGCGGGCCGCCTGTGGCTTCCCGGGTCGCTCCCCCGACTCAGAAAGCTCTCAAGTTGGTATCGTTTTCCCGGCCCTCGGAGGTGGATTGCAGATCACCGAGAGGGGATTTACCAGTAACCACTACAGAATCTACCCGGGCTTTAACAAGCGCTCATTTCTCTCCCTTGTCCTTAGAAAAACTTCGCGCTGGCGTTGATCATATCGTACTTGTAGCGGCAGCTTAGGGGCAGCGGAACTGGTGGGGTTGTGCGTGCAGGGGGAGGCTGTGAGGGAGCCCTGCACTCCGCCCCTCCACCCTTCTGGAGGAGTGGCTTTGTTTCTAAGGGTGCCCCCCCAACCCCCGGGTCCCCACTTCAATGTTTCTGCTCTTTGTCCCACCGCCCGTGAAAGCTCGGCTTTCATTTGGTCGGCGAAGCCTCCGACGCCCCCGAGTCCCACCCTAGCGGGCCGCGCGGCACTGCAGCCGGGGGTTCCTGCGGACTGGCCCGACAGGGTGCGCGGACGGGGACGCGGGCCCCGAGCACCGCGACGCCAGGGTCCTTTGGCAGGGCCCAAGCACCCCT 59 EDGETGGCGGCCGGCGGGCACAGCCGGCTCATTGTTCTGCACTACAACCACTCGGGCCGGCTGGCCGGGCGCGGGGGGCCGGAGGATGGCGGCCTGGGGGCCCTGCGGGGGCTGTCGGTGGCCGCCAGCTGCCTGGTGGTGCTGGAGAACTTGCTGGTGCTGGCGGCCATCACCAGCCACATGCGGTCGCGACGCTGGGTCTACTATTGCCTGGTGAACATCACGCTGAGTGACCTGCTCACGGGCGCGGCCTACCTGGCCAACGTGCTGCTGTCGGGGGCCCGCACCTTCCGTCTGGCGCCCGCCCAGTGGTTCCTACGGGAGGGCCTGCTCTTCACCGCCCTGGCCGCCTCCACCTTCAGCCTGCTCTTCACTGCAGGGGAGCGCTTTGCCACCATGGTGCGGCCGGTGGCCGAGAGCGGGGCCACCAAGACCAGCCGCGTCTACGGCTTCATCGGCCTCTGCTGGCTGCTGGCCGCGCTGCTGGGGATGCTGCCTTTGCTGGGCTGGAACTGCCTGTGCGCCTTTGACCGCTGCTCCAGCCTTCTGCCCCTCTACTCCAAGCGCTACATCCTCTTCTGCCTGGTGATCTTCGCCGGCGTCCTGGCCACCATCATGGGCCTCTATGGGGCCATCTTCCGCCTGGTGCAGGCCAGCGGGCAGAAGGCCCCACGCCCAGCGGCCCGCCGCAAGGCCCGCCGCCTGCTGAAGACGGTGCTGATGATCCTGCTGGCCTTCCTGGTGTGCTGGGGCCCACTCTTCGGGCTGCTGCTGGCCGACGTCTTTGGCTCCAACCTCTGGGCCCAGGAGTACCTGCGGGGCATGGACTGGATCCTGGCCCTGGCCGTCCTCAACTCGGCGGTCAACCCCATCATCTACTCCTTCCGCAGCAGGGAGGTGTGCAGAGCCGTGCTCAGCTTCCTCTGCTGCGGGTGTCTCCGGCTGGGCATGCGAGGGCCCGGGGACTGCCTGGCCCGGGCCGTCGAGGCTCACTCCGGAGCTTCCACCACCGACAGCTCTCTGAGGCCAAGGGACAGCTTTC60 chr13TAGTAAGGCACCGAGGGGTGGCTCCTCTCCCTGCAGCGGCTGTCGCTTACCATCCTGTAGACCGTGACCTCCTCACACAGCGCCAGGACGAGGATgroup-CGCGGTGAGCCAGCAGGTGACTGCGATCCTGGAGCTGGTCGCAGCAGGCCATCCTGCACGCGGTGGAGGCGCCCCCTGCAGGCCGCAGCGCATCC00005CCAGCTTCTGGACGCACTGTGAGCGGTTATGCAGCAGCACGCTCATATGAGATGCCCCGCAGGGTGCTATGCAGGCCCACGTCCCCACAAAGCCCATGGCAGGCGCCCGGGTGCCGGAGCACGCACTTGGCCCCATGGATCTCTGTGCCCAGGGCTCAGCCAGGCATCTGGCCGCTAAAGGTTT61 CRYLTCTCATCTGAGCGCTGTCTTTCACCAGAGCTCTGTAGGACTGAGGCAGTAGCGCTGGCCCGCCTGCGAGAGCCCGACCGTGGACGATGCGTCGCG1CCCTTCCCATCGCGGCCTGGGCGGGCCCGCCTGCCCTCGGCTGAGCCCGGTTTCCCTACCCCGGGGCACCTCCCCTCGCCCGCACCCGGCCCCAGTCCCTCCCAGGCTTGCGGGTAGAGCCTGTCTTTGCCCAGAAGGCCGTCTCCAAGCT 62 IL17CAGTCCCCGAGGCCCTCCCCGGTGACTCTAACCAGGGATTTCAGCGCGCGGCGCGGGGCTGCCCCCAGGCGTGACCTCACCCGTGCTCTCTCCCTDGCAGAATCTCCTACGACCCGGCGAGGTACCCCAGGTACCTGCCTGAAGCCTACTGCCTGTGCCGGGGCTGCCTGACCGGGCTGTTCGGCGAGGAGGACGTGCGCTTCCGCAGCGCCCCTGTCTACAT 63 IRS2AGAGAGACATTTTCCACGGAGGCCGAGTTGTGGCGCTTGGGGTTGTGGGCGAAGGACGGGGACACGGGGGTGACCGTCGTGGTGGAGGAGAAGGTCTCGGAACTGTGGCGGCGGCGGCCCCCCTGCGGGTCTGCGCGGATGACCTTGGCGCCGCGGTGGGGGTCCGGGGGCTGGCTGGCCTGCAGGAAGGCCTCGACTCCCGACACCTGCTCCATGAGGCTCAGCCTCTTCACGCCCGACGTCGGGCTGGCCACGCGGGCAGCTTCTGGCTTCGGGGGGGCCGCGATAGGTTGCGGCGGGGTGGCGGCCACACCAAAAGCCATCTCGGTGTAGTCACCATTGTCCCCGGTGTCCGAGGACAACGATGAGGCGGCGCCCGGGCCCTGGGCGGTGGCAACGGCCGAGGCGGGGGGCAGGCGGTACAGCTCCCCCGGGGCCGGCGGCGGTGGCGGCGGCTGCAGAGACGACGACGGGGACGCGGACGGACGCGGGGGCAACGGCGGATACGGGGAGGAGGCCTCGGGGGACAGGAGGCCGTCCAAGGAGCCCACGGGGTGGCCGCTCGGGGCGCCCGGCTTAGGAGACTTGGGGGAGCTGAAGTCGAGGTTCATGTAGTCGGAGAGCGGAGACCGCTGCCGGCTGTCGCTGCTGGTGCCCGGGGTGCCTGAGCCCAGCGACGAGGCCGGGCTGCTGGCGGACAAGAGCGAGGAGGACGAGGCCGCCGACGCCAGCAGGGGAGGCGCGGGCGGCGACAGGCGGGCCCCGGGCTCGCCAAAGTCGATGTTGATGTACTCGCCGGGGCTCTTGGGCTCCGGTGGCAGTGGGTACTCGTGCATGCTGGGCAGGCTGGGCAGCCCCTCCAGGGACAGGCGCGTGGGCCTCACCGCCCGGCCGCGCTGGCCCAAGAAGCCCTCCGGGCGGCCGCCGCTAGGCCGCACGGGCGAAGGCACTACAGGGTGAGGGGGCTGCGTGGGGCCGGCCCCGAAGGCGCTGGCCGCCTGGCTGGGCCCTGGCGTGGCCTGAGGCTCCAGACGCTCCTCCTCCAGGATGCGCCCCACGGGGGAGCTCATGAGCACGTACTGGTCGCTGTCCCCGCCACAGGTGTAGGGGGCCTTGTAGGAGCGGGGCAAGGAGCTGTAGCAGCAGCCGGGAACGCCCCTGAGCGGCTCCCCGCCGGGGTGCAGGGCTGCGGAGAAGAAGTCGGGCGGGGTGCCCGTGGTGACCGCGTCGCTGGGGGACACGTTGAGGTAGTCCCCGTTGGGCAGCAGCTTGCCATCTGCATGCTCCATGGACAGCTTGGAACCGCACCACATGCGCATGTACCCACTGTCCTCGGGGGAGCTCTCGGCGGGCGAGCTGGCCTTGTAGCCGCCCCCGCTCGCCGGGAATGTCCTGCCCGCCGCAGAGGTGGGTGCTGGCCCCGCAGGCCCCGCAGAAGGCACGGCGGCGGCGGCGGCGGCGGCGGCCCTGGGCTGCAAGATCTGCTTGGGGGCGGACACGCTGGCGGGGCTCATGGGCATGTAGTCGTCGCTCCTGCAGCTGCCGCTCCCACTGCCCGCGAGGGCCGCGCCGGGCGTCATGGGCATGTAGCCGTCGTCTGCCCCCAGGTTGCTGCTGGAGCTCCTGTGGGAGCCGATCTCGATGTCTCCGTAGTCCTCTGGGTAGGGGTGGTAGGCCACCTTGGGAGAGGACGCGGGGCAGGACGGGCAGAGGCGGCCCGCGCTGCCCGAGAAGGTGGCCCGCATCAGGGTGTATTCATCCAGCGAGGCAGAGGAGGGCTGGGGCACCGGCCGCTGCCGGGCTGGCGTGGTCAGGGAGTAGGTCCTCTTGCGCAGCCCTCGGTCCAGGTCCTGGGCCGCGTCCCCCGAGACCCGGCGGTAGGAGCGGCCACAGTGGCTCAGGGGCCTGTCCATGGTCATGTACCCGTAGAACTCACCGCCGCCGCCGCCGTCTCGGGCCGGGGGCGTCTCCGCGATGGACTCGGGCGTGTTGCTTCGGTGGCTGCAGAAGGCGCGCAGGTCGCCTGGGCTGGAGCCGTACTCGTCCAGGGACATGAAGCCGGGGTCGCTGGGGGAGCCCGAGGCGGAGGCGCTGCCGCTGGAGGGCCGCTGGCCGGGGCCGTGGTGCAGCGGATGCGGCAGAGGCGGGTGCGGGCCGGGCGGCGGCGGGTAGGAGCCCGAGCCGTGGCCGCTGCTGGACGACAGGGAGC 64 chr13TAACCTAAAGAATGAAGTCATGCCCCGGCCTGCACCCGGGAAACTGCACACAGCGAAAGATCGCCACTGAGATAAAGAGCTGAAAGCTATTCCCCgroup- AATTCAGCTGTTTCAGCCGTGCGGTCTCACAATGGGCTCACAGACGGCAGCATC 00350 65MCF2LGTTTCCACAATCCACCTCGTAGCTGGGGCGTGCCGCTTGCCTCGGCTTGTCCCGGCAGAACACTCTTACCTTTAATGGCGACTGAAAAGTTGCCACGAGTTCCTGATCATTGTGGTAGGTGCTGCGTGAAGCTGAGACGTGCGTGAGCCACATCCCAGGGGGCTTTGAGCCCCCACCGCGGCGGCGGCTGAGGGGAGGCTTGTCGTACTCGCACAGGAGGACACAGGGCTGCAGTGTTCACTCCAGGGCCTCTTATCATTGGGATCTGAGGAATTTTCCGAGAGGAAGTGCGAATTAACAATGATGAAAGGTTTGTGAGTGAGTGACAGGCACGTTCTATTGAGCACTGCATGGGGCATTATGTGCCACCAGAGACGGGGGCAGAGGTCAAGAGCCCTCGAGGGCTGGGAGAGTTCGGAGGATAGAAGTCATCAGAGCACAATGAAGCCAGACCCTGCAGCCGCCTTCCCCTTCGGGGGCTTCCTTAGAATGCAGCATTGCGGGGACTGAGCTGTCCCAGGTGAAGGGGGGCCGTCACGGTGTGTGGACGCCCCTCGGCTCAGCCCTCTAAGAGACTCGGCAGCCAGGATGGGCTCAAGGCATGAGCCCTCAAAGGAGGTTAGGAAGGAGCGAGGGAGAAAAGATATGCTTGTGTGACGTCCTGGCCGAAGTGAGAACAATTGTATCAGATAATGAGTCATGTCCCATTGAGGGGTGCCGACAAGGACTCGGGAGGAGGCCACGGAGCCCTGTACTGAGGAGACGCCCACAGGGAGCCTCGGGGGCCCAGCGTCCCGGGATCACTGGATGGTAAAGCCGCCCTGCCTGGCGT66 F7TCCAGCTGCAGCGAGGGCGGCCAGGCCCCCTTCTCCGACCTGCAGGGGTAGCGCGGCCTCGGCGCCGGAGACCCGCGCGCTGTCTGGGGCTGCGGTGGCGTGGGGAGGGCGCGGCCCCCGGACGCCCCGAGGAAGGGGCACCTCACCGCCCCCACCCAGAGCGCCTGGCCGTGCGGGCTGCAGAGGACCCCTCCGGGGCAGAGGCAGGTTCCACGGAAGACCCCGGCCCGCTGGGGCTTCCCCGGAGACTCCAGAG 67chr18ACTTACTGCTTCCAAAAGCGCTGGGCACAGCCTTATATGACTGACCCCGCCCCCGAGTCCCAGGCCGCCCCATGCAACCGCCCAACCGCCCAACCgroup-GCCACTCCAAAGGTCACCAACCACTGCTCCAGGCCACGGGCTGCCTCTCCCCACGGCTCTAGGGCCCTTCCCCTCCACCGCAGGCTGAC00039 68 C18TGCCACACCCAGGTACCGCCCGCCCGCGCGAGAGCCGGGCAGGTGGGCCGCGGATGCTCCCAGAGGCCGGCCCAGCAGAGCGATGGACTTGGACAorf1 TGGCAAGATGGAAGTGACCTGAG 69 CD33TCGCCAGCGCAGCGCTGGTCCATGCAGGTGCCACCCGAGGTGAGCGCGGAGGCAGGCGACGCGGCAGTGCTGCCCTGCACCTTCACGCACCCGCAL3CCGCCACTACGACGGGCCGCTGACGGCCATCTGGCGCGCGGGCGAGCCCTATGCGGGCCCGCAGGTGTTCCGCTGCGCTGCGGCGCGGGGCAGCGAGCTCTGCCAGACGGCGCTGAGCCTGCACGGCCGCTTCCGGCTGCTGGGCAACCCGCGCCGCAACGACCTCTCGCTGCGCGTCGAGCGCCTCGCCCTGGCTGACGACCGCCGCTACTTCTGCCGCGTCGAGTTCGCCGGCGACGTCCATGACCGCTACGAGAGCCGCCACGGCGTCCGGCTGCACGTGACAGGCGAGGCGGCGTGGGAGCGGGTCCCCGGCCTCCCTTCCCGCCCTCCCGCCTGCCCCGCCCCAAGGGCTACGTGGGTGCCAGGCGCTGTGCTGAGCCAGGAAGGGCAACGAGACCCAGCCCTCTCCTCTACCCCAGGGATCTCACACCTGGGGGTAGTTTAGGACCACCTGGGAGCTTGACACAAATGCAGAATCCAGGTCCCAGGAAGGGCTGAGGTGGGCCCGGGAATAGGCATTGCCGTGACTCTCGTAGAGTGACTGTCCCCAGTGGCTCTCAGACGAAGAGGCGAGAAAGACAAGTGAATGGCAATCCTAAATATGCCAAGAGGTGCAATGTGGTGTGTGCTACCAGCCCGGAAAGACACTCGCAGCCCCTCTACCCAGGGGTGCACAGACAGCCCACCAAGTAGTGCCTAGCACTTTGCCAGACCCTGATATACAAAGATGCCTGAACCAGGGTCCCGTCCCTAGAGCAGTGGCTCTCCACTCTAGCCCCCACCCTGCTCTGCGACAATAATGGCCACTTAGCATTTGCTAGGGAGCCGGGACCTAGTCCAAGCACCCACAAGCATGAATTTGCCAAATCTTTTCAGCAACCTCTTAAGGCAACTGCTATCATGATCCTCACTTTACACATGGAGAAGCAGAAGCAGAGATGATAGAATCTTTCGCCCAAGGCCACATCTGTATTGGGACGGGGGCAGCCTGGCACCCAAGTGCCCATTCCTCCCTTCTGACCAGCCCCCACCCCTCCGGCTCTGGCGTCCAAAGGGCTAAGGGGAGGGGTGCCCTTGTGACAGTCACCCGCCTTCTCCCCTGCAGCCGCGCCGCGGATCGTCAACATCTCGGTGCTGCCCAGTCCGGCTCACGCCTTCCGCGCGCTCTGCACTGCCGAAGGGGAGCCGCCGCCCGCCCTCGCCTGGTCCGGCCCGGCCCTGGGCAACAGCTTGGCAGCCGTGCGGAGCCCGCGTGAGGGTCACGGCCACCTAGTGACCGCCGAACTGCCCGCACTGACCCATGACGGCCGCTACACGTGTACGGCCGCCAACAGCCTGGGCCGCTCCGAGGCCAGCGTCTACCTGTTCCGCTTCCATGGCGCCAGCGGGGCCTCGACGGTCGCCCTCCTGCTCGGCGCTCTCGGCTTCAAGGCGCT 70 TNFRATGAACTTCAAGGGCGACATCATCGTGGTCTACGTCAGCCAGACCTCGCAGGAGGGCGCGGCGGCGGCTGCGGAGCCCATGGGCCGCCCGGTGCASFGGAGGAGACCCTGGCGCGCCGAGACTCCTTCGCGGGGAACGGCCCGCGCTTCCCGGACCCGTGCGGCGGCCCCGAGGGGCTGCGGGAGCCGGAGA11AAGGCCTCGAGGCCGGTGCAGGAGCAAGGCGGGGCCAAGGCTTGAGCGCCCCCCATGGCTGGGAGCCCGAAGCTCGGAGC71 ZNFTCAGTGTTATGTGGGGAGCGCTAGATCGTGCACACAGTAGGCGTCAGGAAGTGTTTTCCCCAGTAATTTATTCTCCATGGTACTTTGCTAAAGTC236ATGAAATAACTCAGATTTTGTTTTCCAAGGAAGGAGAAAGGCCCAGAATTTAAGAGCAGGCAGACACACAACCGGGCACCCCCAGACCCTGGCCCTTCCAGCAGTCAGGAATTGACTTGCCTTCCAAAGCCCCAGCCCGGAGCTTGAGGAACGGACTTTCCTGCGCAGGGGGATCGGGGCGCACTCG72 chr18GTGGAAACACAACCTGCCTTCCATTGTCTGCGCCTCCAAAACACACCCCCCGCGCATCCGTGAAGCTGTGTGTTTCTGTGTTACTACAGGGGCCGgroup- GCTGTGGAAATCCCACGCTCCAGACCGCGTGCCGGGCAGGCCCAGCC 00342 73 OLIG2TCCACACCTCGGGCAGTCACTAGGAAAAGGGTCGCCAACTGAAAGGCCTGCAGGAACCAGGATGATACCTGCGTCAGTCCCGCGGCTGCTGCGAGTGCGCGCTCTCCTGCCAGGGGGACCTCAGACCCTCCTTTACAGCACACCGAGGGCCCTGCAGACACGCGAGCGGGCCTTCAGTTTGCAAACCCTGAAAGCGGGCGCGGTCCACCAGGACGATCTGGCAGGGCTCTGGGTGAGGAGGCCGCGTCTTTATTTGGGGTCCTCGGGCAGCCACGTTGCAGCTCTGGGGGAAGACTGCTTAAGGAACCCGCTCTGAACTGCGCGCTGGTGTCCTCTCCGGCCCTCGCTTCCCCGACCCCGCACAGGCTAACGGGAGACGCGCAGGCCCACCCCACCGGCTGGAGACCCCGGCACGGCCCGCATCCGCCAGGATTGAAGCAGCTGGCTTGGACGCGCGCAGTTTTCCTTTGGCGACATTGCAGCGTCGGTGCGGCCACAATCCGTCCACTGGTTGTGGGAACGGTTGGAGGTCCCCCAAGAAGGAGACACGCAGAGCTCTCCAGAACCGCCTACATGCGCATGGGGCCCAAACAGCCTCCCAAGGAGCACCCAGGTCCATGCACCCGAGCCCAAAATCACAGACCCGCTACGGGCTTTTGCACATCAGCTCCAAACACCTGAGTCCACGTGCACAGGCTCTCGCACAGGGGACTCACGCACCTGAGTTCGCGCTCACAGATC74 RUNX1CTGCCCTCGCGGATCTCCCCCGGCCTCGCCGGCCTCCGCCTGTCCTCCCACCACCCTCTCCGGGCCAGTACCTTGAAAGCGATGGGCAGGGTCTTGTTGCAGCGCCAGTGCGTAGGCAGCACGGAGCAGAGGAAGTTGGGGCTGTCGGTGCGCACCAGCTCGCCCGGGTGGTCGGCCAGCACCTCCACCATGCTGCGGTCGCCGCTCCTCAGCTTGCCGGCCAGGGCAGCGCCGGCGTCCGGGGCGCCCAGCGGCAACGCCTCGCTCATCTTGCCTGGGCTCAGCGCGGTGGAAGGCGGCGTGAAGCGGCGGCTCGTGCTGGCATCTACGGGGATACGCATCACAACAAGCCGATTGAGTTAGGACCCTGCAAACAGCTCCTACCAGACGGCGACAGGGGCGCGGATCTTCAGCAAGCAGCTCCCGGGAGACCAACATACACGTTCAGGGGCCTTTATTACTGCGGGGGGTGGGGGGGGGCGGGGGTGGTTAGGGGAGGAGGGAGACTAAGTTACTAACAGTCCAGGAGGGGAAAACGTTCTGGTTCTGCGGATCGGCCTCTGACCCAGGATGGGCTCCTAGCAACCGATTGCTTAGTGCATTAAAAAGTGGAGACTATCTTCCACGAATCTTGCTTGCAGAGGTTAAGTTCTGTCTTTGGCTGTTAGAAAAGTTCCTGAAGGCAAAATTCTCATACACTTCCTAAAATATTTATGCGAAGAGTAAAACGATCAGCAAACACATTATTTGGAAGTTCCAGTAGTTAATGCCTGTCAGTTTTTTGCAGGTGAGTTTTGTCTAAAGTCCCAACAGAACACAATTATCTCCCGTAACAAGGCCACTTTTATCATGCAAAACTGGCTTCAGTCCCGAAAAGCAAGAGCTGAGACTTCCAAAGGTAGTGCTACTAATGTATGTGCACGTATATATAAATATATACATATGCTCTACTTCATAAAATATTTACAATACAATCTGTGGAGAATTTAAACACAACAGAAATCCATTAATGTACGCTGCAGATTTTTTTAAGTAGCCTTGAAAATCAGCTTCAGTAGTTGGAGCAGTGCTGAGCTAGAAGTACTTGTCATGTTCTCTGTTCTCTCAATGAATTCTGTCAAAACGCTCAGTGCAGAAAATTCAGCGTTTCAGAGATCTTCAGCTAATCTTAAAACAACAATCATAAGAAGGCCCAGTCGATGACACTCAGGGTTCTACAGCTCTCCCACATCTGTGAACTCGGGTTTGGGGATGTTGGTTAAGTTTGTGGCTGGTCCTCTGGTTTGTTGGGAGTTGAGCAGCCGCAGAGTCACACACATGCAAACACGCACTCTTCGGAAGGCAGCCACTGTCTACATCAGCTGGGTGACTCAGCCCTGACTCGGGCAGCAGCGAGACGATACTCCTCCACCGTCGCCCAGCACCACGCCGGTTAGCTGCTCCGAGGCACGAACACCCACGAGCGCCGCGTACCGCAGCAGGTGGAGCGGGCCTTGAGGGAGGGCTCCGCGGCGCAGATCGAAACAGATCGGGCGGCTCGGGTTACACACGCACGCACATCCTGCCACGCACACTGCCACGCACACGCAACTTCACGGCTCGCCTCGGACCACAGAGCACTTTCTCCCCCTGTTGTAAAAGGAAAACAATTGGGGAAAAGTTCGCAGCCAGGAAAGAAGTTGAAAACATCCAGCCAAGAAGCCAGTTAATTCAAAAGGAAGAAAGGGGAAAAACAAAAAAAAACAACAAAAAAAGGAAGGTCCAACGCAGGCCAAGGAGAAGCAGCAGAGGTTGACTTCCTTCTGGCGTCCCTAGGAGCCCCGGAAAGAAGTGCCTGGCGGCGCAGGGCCGGGCAGCGTGGTGCCCTGGCTGGGTCCGGCCGCGGGGCGCCCGTCCCGCCCGCGCCCGCTGGCTCTATGAATGAGAGTGCCTGGAAATGAACGTGCTTTTACTGTAAGCCCGGCCGGAGGAATTCCATTCCCTCAGCTCGTTTGCATAGGGGCGGCCGGCGGCCAATCACAGGCCTTTCCGGTATCAGCCAGGGCGCGGCTCGCCGCCGCCGGCTCCTGGAATTGGCCCGCGCGCCCCCGCCGCCGCGCCGCGCGCTACTGTACGCAGCCCGGGCGGGGAGTCGGAGGCCACCCCCGCGCCCCGCATCCAAGCCTGCATGCTGGCCCGGGGCCCCGCCCGCGTGCGGACCCCTTTCCGCAGCCACACGCAGGCTTGTGCGGCTCCGCGAGTGGCCACGGTCCGGAGACCTGGAAAAAGAAAGCAGGCCCCGCCGGCCCGAGGAGGACCCGGCCGGCGCGCCGCACCCGGAGAGGCCCGGCCCCGCGAGCCGCTGCAGGCAGGCGCAGTGGCCGCCACGAGGCTCCCGAACCGGGCTGCAGCCCGCGGACGGCCCCAGATCCTGCGCGGCCGCCCAGGGCCAGGCCTCCGCTTCCAGGGCGGGGGTGCGATTTGGCCGCGGGGCCCGGGGGAGCCACTCCGCGCTCCTGCACCGTCCGGCTGGCAGCTGCGGCGAAGCGGCGCTGATTCCTTGCATGAGGCCGGACGGCGTCCGCGCGTGCCGTTTGCTCTCAGCGTCTTCCCTTGGGTCGGTTTCTGTAATGGGTGTTTTTTACCGCTGCGCCCGGGCCGCGGCTCGATCCCTCCGCGCGTCTCACTTGCTGCGTGCGTCAGCGGCCAGCGAAGAGTTTCCTAGTCAGGAAAGACCCCAAGAACGCGCGGCTGGAAGGAAAGTTGAAAGCAGCCACGCGGCTTGCTCCCGGGCCTTGTAGCGCCGGCACCCGCAGCAGCCGGACAGCCTGCCCGGGCCCCGCGTCTCCCCTCCGGCTCCCCGGAAGCGGCCCCCGCTCCTCTCCCCGCCCCCGTGCGCTCGAGCGGCCCCAGGTGCGGAACCCACCCCGGCTTCGCGTGCGGGCGGCCGCTTCCCCCTGCGCCGGTCCCCGCGGTGCTGCGGGCATTTTCGCGGAGCTCGGAGGGCCCCGCCCCCGGTCCGGCGTGCGCTGCCAACTCCGACCCCGCCCGGCGGGGCTCCCTCCCAGCGGAGGCTGCTCCCGTCACCATGAGTCCCTCCACGCCCTCCCTGCCGGGCCCTGCACCTCCCGGGGCCTCTCATCCACCCCGGGGCTGCAACCCAGTCCCCGGATCCCGGCCCCGTTCCACCGCGGGCTGCTTTGTGGTCCCCGCGGAGCCCCTCAATTAAGCTCCCCGGCGCGGGGGTCCCTCGCCGACCTCACGGGGCCCCTGACGCCCGCTCCTCCCTCCCCCAGGGCTAGGGTGCTGTGGCCGCTGCCGCGCAGGGACTGTCCCCGGGCGTTGCCGCGGGCCCGGACGCAGGAGGGGGCCGGGGTTGACTGGCGTGGAGGCCTTTCCCGGGCGGGCCCGGACTGCGCGGAGCTGTCGGGACGCGCCGCGGGCTCTGGCGGACGCCAGGGGGCAGCAGCCGCCCTCCCTGGACGCCGCGCGCAGTCCCCGGAGCTCCCGGAACGCCCCCGACGGCGCGGGGCTGTGCGGCCCGCCTCGTGGCCTTCGGGTCGCCCGGGAAGAACTAGCGTTCGAGGATAAAAGACAGGAAGCCGCCCCAGAGCCCACTTGAGCTGGAACGGCCAAGGCGCGTTTCCGAGGTTCCAATATAGAGTCGCAGCCGGCCAGGTGGGGACTCTCGGACCAGGCCTCCCCGCTGTGCGGCCCGGTCGGGGTCTCTTCCCGAAGCCCCTGTTCCTGGGGCTTGACTCGGGCCGCTCTTGGCTATCTGTGCTTCAGGAGCCCGGGCTTCCGGGGGGCTAAGGCGGGCGGCCCGCGGCCTCAACCCTCTCCGCCTCCGCTCCCCCTGGGCACTGCCAGCACCCGAGTTCAGTTTTGTTTTAATGGACCTGGGGTCTCGGAAAGAAAACTTACTACATTTTTCTTTTAAAATGATTTTTTTAAGCCTAATTCCAGTTGTAAATCCCCCCCTCCCCCCGCCCAAACGTCCACTTTCTAACTCTGTCCCTGAGAAGAGTGCATCGCGCGCGCCCGCCCGCCCGCAGGGGCCGCAGCGCCTTTGCCTGCGGGTTCGGACGCGGCCCGCTCTAGAGGCAAGTTCTGGGCAAGGGAAACCTTTTCGCCTGGTCTCCAATGCATTTCCCCGAGATCCCACCCAGGGCTCCTGGGGCCACCCCCACGTGCATCCCCCGGAACCCCCGAGATGCGGGAGGGAGCACGAGGGTGTGGCGGCTCCAAAAGTAGGCTTTTGACTCCAGGGGAAATAGCAGACTCGGGTGATTTGCCCCTCGGAAAGGTCCAGGGAGGCTCCTCTGGGTCTCGGGCCGCTTGCCTAAAACCCTAAACCCCGCGACGGGGGCTGCGAGTCGGACTCGGGCTGCGGTCTCCCAGGAGGGAGTCAAGTTCCTTTATCGAGTAAGGAAAGTTGGTCCCAGCCTTGCATGCACCGAGTTTAGCCGTCAGAGGCAGCGTCGTGGGAGCTGCTCAGCTAGGAGTTTCAACCGATAAA 75 AIRETTCGGAAGTGAGAGTTCTCTGAGTCCCGCACAGAGCGAGTCTCTGTCCCCAGCCCCCAAGGCAGCTGCCCTGGTGGGTGAGTCAGGCCAGGCCCGGAGACTTCCCGAGAGCGAGGGAGGGACAGCAGCGCCTCCATCACAGGGAAGTGTCCCTGCGGGAGGCCCTGGCCCTGATTGGGCGCCGGGGCGGAGCGGCCTTTGCTCTTTGCGTGGTCGCGGGGGTATAACAGCGGCGCGCGTGGCTCGCAGACCGGGGAGACGGGCGGGCGCACAGCCGGCGCGGAGGCCCCACAGCCCCGCCGGGACCCGAGGCCAAGCGAGGGGCTGCCAGTGTCCCGGGACCCACCGCGTCCGCCCCAGCCCCGGGTCCCCGCGCCCACCCCATGGCGACGGACGCGGCGCTACGCCGGCTTCTGAGGCTGCACCGCACGGAGATCGCGGTGGCCGTGGACAG76 SUMO3ACGCACACTGGGGGTGTGATGGAAAGGGGGACGCGATGGATAGGGGTGGGCGCACACTGGGGGACGCGACGGGGAGGGGTGAGCACACACTGGGGGTGTGATGGAGAGGGCGACGCAATAGGGAGGGGTGGGCGCACACCAGGGACGCGATGATGGGGACGGGTGGGCGCACACCAGGTGGCATGATGGGGAGGAGTGGGTACACACCATGGGGGGCGTGATGGGGAGGCGTGGGCGTACACCGGGGGGCGCGATGGGGAGGGGTGGGCGCACACCGGGGGACGCGATGGAGGCGGTGGGTGCACACGGGGCGCGATGGGTGGGAGTAGGTGCACACTGAGGGCACGATTGGGGAGACACGAAGGAGAGGGGTGGGCGCACACTGGGGGACGCGATGGCCGGGACACGATGCGGAGAAGTGGGTGAATACCGGGGTCGCGATGGGCGCCCTGGAAGGACGGCAGTGCTGCTCACAGGGGCCAGGCCCCTCAGAGCGCGCCCCTTGGGGGTAACCCCAGACGCTTGTTCCCGAGCCGACTCCGTGCACTCGACACAGGATC77 C21CCACAGGGTGGGGTGCGCCCACCTGCCCTGTCCATGTGGCCTTGGGCCTGCGGGGGAGAGGGAATCAGGACCCACAGGGCGAGCCCCCTCCGTAGorf70 CCCGCGGCACCGACTGGATCTCAGTGAACACCCGTCAGCCCATCCAGAGGCTAGAAGGGGGA 78C21orTTGAGGTCTCTGTGCATGCTTGTGCGTACCCTGGACTTTGCCGTGAGGGGTGGCCAGTGCTCTGGGTGCCTTTGCCAGACAACTGGTCTGCCGGGf123 CCGAGCATTCATGCTGGTC 79 COL18TGACGCGCCCCTCTCCCCGCAGCTCCACCTGGTTGCGCTCAACAGCCCCCTGTCAGGCGGCATGCGGGGCATCCGCGGGGCCGACTTCCAGTGCTAl TCCAGCAGG 80 PRRT3AACACACTGTCTCGCACTAGGTGCTCGCGGAAGAGCGCGGCGTCGATGCTGCGGCTCAGGTTGATGGGCGATGGCGGCCGCAGATCCAGCTCGCTCAGCGATGGCGCCGGTCCCACACCGTTGCGGGACAGTCCCGGGCCACCCTGGGGTCCGCGACCCAACGACGCAGCCGAGCCCCAGGCGCCTGAACTGGGCGTGGCCAGCTGCCCACTCTCCGCCGGGTTGCGGATGAGGCTCTTGCTGATGTCCAAGCTGCCTGCACCAACGTTGCTGGGCCCTGCATAGCAGTTATTGGGTCGCTCCGGCACCTCGCTCTTTCCTGACGGCGCCGGGCACGCCAGACGCATCAGCTTAGCCCAGCAAGCGTGCTCCGTGGGCGGCCTGGGTCTCGCGGCAGCCACCGCGGCCAACGCCAGGGCGAGCGCCCATGTCAGCTCCAGGAGGCGCAGCCAGAAGTGGACACCCCACCAGGCCCACGAGAAGCGGCCCACGCGGCCTGGGCCCGGGTACAGCCAGAGCGCAGCCGCCAGCTGCAAGCCGCTAGCCAGCAGCCCCAGCGCGCCCGCCACAGCCAACAGCCGAGGGCCCGGGCTGGCATCCCAGCCCCGTGGGCCGTCCAGCAGGCGGCGACGGCACAGGCAGAGCGTGCCCAGAGCCAC81 MGCGTCTGCACGAAGCCCGCGGCGGCCTGCAGGGGGCCCAGCGACTCGTCCAGGGAACCGGTGCGCAGGAGCAGCCGGGGGCGCGGCGCGCCGGCCGC29506CCTTGGGGGACTCTGGGGCCGGGGGCGCAGCTCGATCTGACGCTTGGGCACTGTCCGGGGCCTGGCGGGCGCGGCGCCCTCCTCCAGAGCCACCTCCACACACTCGAACTGCGCTGGGGCGGCAGGACTTGGCCCACGGGGCCGCAGCTCTAGGTAGGTGGCCCAGCGGGAGCCACCATCGGGGACCTGGGACTGGCGTGGGACCGCGGCGGGAGACGCTGGCCCCGGCGGCAAGGGGCTGATGAAGGCCGGCTCCGTGAACTGTTGTTGCGCCTCGCGATCGTCTGCGCCGGAGCAGCCGAACAGGGGTCCGACGCCGAAGATGACTTCCATCTCCCCCGACGGCAGCGTGCGCAGCTGGGGCTGGGGTGGCCGTGGGCCGGAACCTGGGCCTCGCGGGAAACCCGAGCCGGGCCCGTGCCGCTGGCGGCTATTCTGGGCGCTGACGGACAGGCGAGGCTGCGCGCCCGCCCCCCGCCCAGGAGCCACCCAGGGCCAATTCGCTGGGCCTTTCGCGTCCGGCCCAACGTCCGGGGGCTCCGGAGAACCTGGAGCCGTGTAGTAGGAGCCTGACGAACCGGAGGAGTCCTGGCGCCGCGCGGGGGCCGTGGGCAGCTGCCTCGGGATCCCAGGCAGGGCTGGCGGGGCGAGCGCGGTCAGCATGGTGGGGCCGGACGCCGTGCACTATCTCCCTCGCATTCGCCTCCGCTGGTGGCGC 82 TEAD3CTGGAGAGAACTATACGGGCTGTGGGAGTCACCGGGCGACTATCACCGGGCCTCCTTTCCACATCCTCCTCCGGGAAGGGACCCCGTTCCGGGCCTCGACCGGCGCAGACTGGGCTGACCCACTTTCTTGGGCCCACTGAGTCACCTCGAAACCTCCAGGCCGGTAGCGGGGAGGAGAGGAGGAGCAGGCGGGGGTGCCAAGGTGTGGGCTGCGCCCTGGTTAGGGGGCGAGCCCGGCTTGTTTATGAGGAGGAGCGCGGAGGAGGATCCAGACACACAGGCTTGCGCGCCCAGACTCGCCCGGCCAGCGGCTGGCGGCCTCCGACGTCACCAAACCGGTTGGGTGAGAGGGCAGAGAGCAGGGGGAAGGGCCGCAGTCCCGCCCGCGCCCCCCGGCACGCACCGTACATCTTGCCCTCGTCTGACAGGATGATCTTCCG 83 chr12GAGTGCGGAGTGAAGGGGTGCACTGGGCACTCAGCGCGGCCCTTGGGAGGCAGGGCCGCCCCAGCCTGCCCTCCTGTCTGGGAAGGCCGTCCAGAgroup-AGCAGGAGCCCCGGGGAAAACAACTGGCTGGACGGGGCGGCCTTCAGTGTCTCTCCCAGCCTGAGAGTCGCTTCCCACCACCTGGGCACGAACCT00022 GCTCTGCGATCTCCGGCAAGTTCCTGCGCCTCCTGTCGGTAAAATGCAGATCGTGGCGTCTT 84CENTTCTTCTTTCCGCCCCTAGGGGGCACAAGCGGGCATGTCCAAGCGCCTAGGAGCCCGTACCGCTGGGGACCTCCCCTTCCGCGAACCCCGAGCGGGG1TAGACCCAGAGCAATCCGAGTGTGGAAACAATGGAGAGGGGGCGTGTTGAGCTGGGGTCTCCATGCCTCGTTGGGGAGAGGGAGGTGAGTTTGTGTCTTCTGGAAGGCGTGGGGGCTGTGCCCTCGTGGGGGTAGGAAGTGCTCCCGTGGGGCGGGGTGCGGATCGGAGAGGTGAGTGGGTGCGTCTGTCCAGCGGTCCGCCCGGTGTGGTCGTGCCCGGCCCGCGTGGGGATGGGGGTGTCTCTCCCGCTGGGCAACTATACCAGCGCAACCGGGGCGTCGGCGCGGCCCACGCTAGCGGCGCTGCTCCGGCGGCGGGGGCTGGGCGTGGCGGTGATGCTGGGCGTGGTGGCCGCGCTGGGCGTGGTGGCCGCGCTGCCGCCCTCACCCGGGCAGCCGTGCTGGAGAAGGATGTCGGCGCACAGCTGGCTTCCAGCCTGGCGGGCGTAGAACAGCGCCGTGCGGCCCTGGGCGTCACGGGCCGCCACGTCCGCGCCGTACTAGAGGGCGGAAACGGCCGCGTGACCGCGCGTCCCCAGGGCGCCCACACCCGGCGCCGCCTCCCCCACATGGCCAAGCCTACTTCCGGGGTCCCTCTGGGAATTTCGGGCTTTCCCGCGCCAGGCGTTTTCCGAGATGAAGCCTCAAAGACCCCCTTTCCTCCCCCCAGCTCACGTACCCACAGCAGCAGTTGCGTGATGACGACGTGGGCGAGCTCGGCCGCCAGGTGGAGTGGGGAGCGCAGCTGTGGGTCCTCTACGCTGGTGTCGAGCGGCCCGTGTCGCGCATGGGCCAAAAGCAGGAGAACGGTAGCCACGTCCTGGGCCTGCACGGCGGCCCACAGCTGGCGGCCCAGCGGCTCCTCCGAGGTGCTCAGCGGCGCCAGGAACAGTAGCTGCTCGTACTTGGCGCGAATCCACGACTCGCGCTCCTCCCTGCAAGACCAGGGATCAACGGAAAAGGCTCTAGGGACCCCCAGCCAGGACTTCTGCCCCTACCCACGGGACCGTCTCAGGTTCGCACACCCTCAGCAACCCTCCCCCCGCTCTGTTCCCTCACGCTTACCGCGAAGAGTCCCGCGAGGGCTTGGCACGGCCTCGCGTGTCGCTTTCCCACACGCGGTTGGCCGTGTCGTTGCCAATAGCCGTCAGCACCAGGGTCAGCTCCCGTGGCCAGTCGTCCAAGTCCAGCGAGCGAACGCGGGACAGGTGTGTGCCCAGGTTGCGGTGGATGCCAGAACACTCGATGCAGATGAGGGCGCCCAGGTTCAAGCTGGCCCACGTGGGGTCTGCGGAAGGAGCGTAGAGGTCGGCTCCCAGCCGGGCAGCACAGGCACCCCGGCATTCACTACACTCCCTAGCCCCTCCGCTGCCTCCTGGCACTCACTGGGGGCCCCGCAGTCCACGCAGATTGAATTCCCCTTGGCGTTCCGGATCGCCTGGAT 85 CENTAGCCAGGTCCAGCCCCCGCGCCTGACACCGGCCGGACGTTCCCGGGGCGCCGCAGCTGCGGCGGGAACTCTGGGATCCGGAGCCATCTGCTCCCAG1CCCGCTCCGGAGCCAAACCCCGGGGGCCGCCTCCGCTCCCGGACCCGCCTCCTCTCCCGGGAGTGTGAGCCGAACCAAGAGTCTCCTGCCTATCTCCTCCAGTAGGAAAATAGTAATAATAATAGACACCCTGCCCCCGTAAAAAACACTACCTTCCCCGTACCGCCTCCCAAGTCTCCCGGGGTACGGATTGCCTTTGCAGCAGTTCCGCCCCACCTGACTCACTCCAGGGTCAGCCCCGGGTGGGTTTCAATGCGGCTCTGGGGAGGGGGTGGGCAGTGGGGGAAGTGAGGCTTCCTATCCGCCCCCTCTCACTTCACATTTAAATATTCTGCACGTTCCAGCCCCCGCGGACTCGCGTACCGCCCAATCCGCCTTCACCGCACGAAAAACATCACTAGCCTGCTCTCAGCCCAGGGGACGACTAGTCCCTGGCGAGAAGCTGCCTGCAAGGTCACTGTCATGCCACCTGCCCCAAGTGCTCAGGGGAAACTGAGGCTTCCTCATCCCCTTCACCTTCAACGTCGCTCTAAACACGGCAAAGCCCCGTTTCCATGCTCCCAGAGTTCAGCTGAGGCTGGAAGTGGGGTCCTGGGCTTCTCTGGGAGCAATTTTCTAGTCACTCTGATCAAGGACGTTACTTTCCCAGAAAGCTCTGAGGCTGAGTCCCTCTGAAATCAAGTCCTTTCTCCTGTCGCACAATGTAGCTACTCGCCCCGCTTCAGGACTCCTATTCTTTGCCCCAATCCTTGACAGAGGGGTGAGCTTGGTTCATCCGCCCACCCCAGAGAAAAGCTTCCCTAGTTTCCTGGACCTCGCTCCTCCACCCCAAGCTGAGCATTCCAGGTACCCTTCCCTCCCTGTTCTCAAGCCCTGACTCAACTCACTAGGGGAAGCGCGGAGCTCGGCGCCCAGCAGCTCCCTGGACCCGCTGCCAGAAGACAGGCTGGGGGGTCCGGGAAGGGGCCCGGAGCCAGGAGGCCCTCCTGTGCTCTTGGTGAAGATGCCGCTGATAAACTTGAGCATCTTGCGGTCACGAGTGGATGCTCGGCCCCCCTCCCGGCCCCGTTTCAGCCCCGGAGCTGGAGGCTCCAGAGTGATTGGAGGTGCAGGCCCGGGGGGCTGCGCGGAAGCAGCGGTGACAGCAGTGGCTGGACTCGGAGTTGGTGGGAGGGTTAGCGGAGGAGGAGAGCCGGCAGGCGGTCCCGGATGCAAGTCACTGTTGTCCAAGGTCTTACTCTTGCCTTTCCGAGGGGACAACTTCCCTCGGGCTCCAGCCCCAGCCCCGACCCCACCAGAGGTCGAAGCTGTAGAGCCCCCTCCCCCGGCGGCGGCGGCGGTGGCGGCGGCAGAGACCGAAGCTCCAGTCCCGGCGCTGCTCTTTGACCCCTTGACCCTGGGCTTGCCCTCGCTTTCGGGCCATGACAGGCGGCTACCCGCGCCCTTGCCCCCGCCGGCTTTGGCTCCACTCGTGGTCACGGTCTTGCAAGGCTTGGGAGCCGGCGGAGGAGGCGCCACCTTGAGCCTCCGGCTGCCGGTGCCAGGGTGCGGAGAGGATGAGCCAGGGATGCCGCCGCCCGCCCGGCCTTCGGGCTCCGGGCCGCCCCAGCTCGGGCTGCTGAGCAGGGGGCGCCGGGAGGAGGTGGGGGCGCCCCCAGGCTTGGGGTCGGGGCTCAGTCCCCCGGAGAGCGGGGGTCCCGGAGGGACGGCCCAGAGGGAGAGGCGGCGGCCGGGAGCGGGGGAGACTGGGCGGGCCGGACTGGCCGGAGCCGGGGACAGGGCTGGGGGCTCCGCGCCCCCGGTGCCCGCGCTGCTCGTGCTGATCCACAGCGCATCCTGCCGGTGGAAGAGACGTTCGTGCCGCTTCTTGCCCGGCTCCTCCGCGCCTCGGGGGCTGCCAGGATCCCCAGTCTCGGAGCCTCTGGCACCGGCGGCGCCGGCCGCGGCCGCAGACGGAGAAGGCGGCGGCGGAGGCACCGACTCGAGCTTAACCAGGGTCAGCGAGATGAGGTAGGTCGTTGTCCGGCGCTGAAGCGCGCCCGCGCCCCGGCTCATGGGGCCCGGAGACCCCCGAGCTGGGGAGGGGAGGGGACTCCCCCGGACTGCCTCAGGGGGGCCCGGCCATGGGGCCGCCCTGCTCGCTGCCCCCAGCCCCCGGACCCCGCTGAGCCCCCGGCCCGGCTCCGCTGTCGCCGCCGCCTCCGCCGCCTCCGCTTGCGCCCCCCTCCCATCACATGGGGCGCCCCCTCCCCATGCTCCCCGCCCTGCGCCCCCACCCTCTTGGAGCCCCGGGACCTTGGTGCTGCTCCAGGGAGGCGCGCCGGACCGTCCACCCCGGCCTGGGTGGGGGCGCTGAGATGGGTGGGGGAGGGCGGGGAGGACAGTAGTGGGGGCAAATGGGGGAGAGAGAGGAAAAGGGAGCAGAAAAGGGGACCGGAGGCTAGGGGAAACGAACCTGTGCGGGGGAGGCAGGGGCGGGGAATTGGGACTCAAGGGACAGGGGCCGCGGATGCGGTCGGAAAGAGGGTCTAGAGGAGGGTGGGAAGCTAGTGG86 chr18AGGAGCGCAAGGCTTGCAGGGCATGCTGGGAGAGCGCAGGGAACGCTGGGAGAGCGCGGGAAATACTGGGATTGGCTCCCGAGGGCTGTGAGGAGgroup-GGCACGAGGGGACACTCCGATGAAGGCAGGGCACGCGGGGCGAGCCGGGAGCGTCTCCTGAGGGCAGCGAGGAGGGAGCTGAGGCACGCGGGCTC00304TCAATCGACGCCCCACAGAGACCAAGAGGCCTGGCCTTGGGGGGCAGCTGCTTGAAGGAGGCAGAGCGGAAGCGAGGGAGACTGCTGGAGGCCCTGCCGCCCACCCGCCCTTTCCTCCCCCTGAGGAGACGCCTGACGCATCTGCAGTGCAGGAGGCCGTGGGCGTTAGAAGTGTTGCTTTTCCAGTTTGTAAGACCATTTTCCTGATTCTCTTCCCCACGGTTGCGGAGGAGCAGGTCAGGGCCGCCATGAGGGCAGGATC87 TSHZ1TCGACCGCTACTATTATGAAAACAGCGACCAGCCCATTGACTTAACCAAGTCCAAGAACAAGCCGCTGGTGTCCAGCGTGGCTGATTCGGTGGCATCACCTCTGCGGGAGAGCGCACTCATGGACATCTCCGACATGGTGAAAAACCTCACAGGCCGCCTGACGCCCAAGTCCTCCACGCCCTCCACAGTTTCAGAGAAGTCCGATGCTGATGGCAGCAGCTTTGAGGAGGC 88 CTDP1TGTGCCGTCGCACACAGACGCCCTCAACGTCGGAGAGCTGTGAGCGGGGCCGTGCTCTTGGGATGGGAGCCCCCGGGAGAGCTGCCCGCCAACACCACTCCGACGTGATCCATGCTGGACATAAAGTGCTCTTCCCTCCGCTAGTCATCGGCCGAGCGGGCCCCTCGCTCCTGGGTGTAAGTTCTTTCTGTGCGTCCTTCTCCCATCTCCGTGCAGTTCAG 89 KCNG2CCATGCGCCGCTGCGCGCGCGAGTTCGGGCTGCTGCTGCTGTTCCTCTGCGTGGCCATGGCGCTCTTCGCGCCACTGGTGCACCTGGCCGAGCGCGAGCTGGGCGCGCGCCGCGACTTCTCCAGCGTGCCCGCCAGCTATTGGTGGGCCGTCATCTCCATGACCACCGTGGGCTACGGCGACATGGTCCCGCGCAGCCTGCCCGGGCAGGTGGTGGCGCTCAGCAGCATCCTCAGCGGCATCCTGCTCATGGCCTTCCCGGTCACCTCCATCTTCCACACCTTTTCGCGCTCCTACTCCGAGCTCAAGGAGCAGCAGCAGCGCGCGGCCAGCCCCGAGCCGGCCCTGCAGGAGGACAGCACGCACTCGGCCACAGCCACCGAGGACAGCTCGCAGGGCCCCGACAGCGCGGGCCTGGCCGACGACTCCGCGGATGCGCTGTGGGTGCGGGCAGGGCGCTGACGCCTGCGCCGCCCAC

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

Modifications may be made to the foregoing without departing from thebasic aspects of the invention. Although the invention has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, yet these modifications and improvements are within thescope and spirit of the invention.

The invention illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and use of such terms and expressions do not exclude anyequivalents of the features shown and described or portions thereof, andvarious modifications are possible within the scope of the inventionclaimed. The term “a” or “an” can refer to one of or a plurality of theelements it modifies (e.g., “a reagent” can mean one or more reagents)unless it is contextually clear either one of the elements or more thanone of the elements is described. The term “about” as used herein refersto a value within 10% of the underlying parameter (i.e., plus or minus10%), and use of the term “about” at the beginning of a string of valuesmodifies each of the values (i.e., “about 1, 2 and 3” refers to about 1,about 2 and about 3). For example, a weight of “about 100 grams” caninclude weights between 90 grams and 110 grams. Further, when a listingof values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or86%) the listing includes all intermediate and fractional values thereof(e.g., 54%, 85.4%). Thus, it should be understood that although thepresent invention has been specifically disclosed by representativeembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and such modifications and variations are considered within thescope of this invention.

Certain embodiments of the invention are set forth in the claims thatfollow.

What is claimed is:
 1. A method for determining fetal fraction in asample comprising: (a) enriching a sample nucleic acid for one or morepolymorphic nucleic acid targets, which sample nucleic acid comprisesfetal nucleic acid and maternal nucleic acid; (b) obtaining nucleotidesequences for some or all of the nucleic acid targets by a sequencingprocess; (c) analyzing the nucleotide sequences of (b); and (d)determining fetal fraction based on the analysis in (c).
 2. The methodof claim 1, wherein the enriching comprises amplifying a plurality ofpolymorphic nucleic acid targets.
 3. The method of claim 1, wherein theenriching comprises generating amplification products in anamplification reaction.
 4. The method of claim 3, wherein theamplification reaction is performed in a single well.
 5. The method ofclaim 1, wherein the polymorphic nucleic acid targets each comprise atleast one single nucleotide polymorphism (SNP).
 6. The method of claim1, wherein the polymorphic nucleic acid targets each comprise at leastone short tandem repeat (STR).
 7. The method of claim 1, whereindetermining the fetal fraction comprises (i) identifying at least oneinformative polymorphic site from the nucleotide sequences in (b), andcalculating the fetal fraction from the amount of fetal polymorphicsites and maternal polymorphic sites.
 8. The method of claim 1, whereinthe maternal genotype at one or more polymorphic nucleic acid targets isnot known prior to (a).
 9. The method of claim 1, wherein 10 or morepolymorphic nucleic acid targets are enriched.
 10. The method of claim1, wherein 50 or more polymorphic nucleic acid targets are enriched. 11.The method of claim 1, wherein 100 or more polymorphic nucleic acidtargets are enriched.
 12. The method of claim 1, wherein 500 or morepolymorphic nucleic acid targets are enriched.
 13. The method of claim1, wherein the sequencing process comprises a sequencing by synthesismethod.
 14. The method of claim 1, wherein the sequencing processcomprises a sequencing by ligation method.
 15. The method of claim 1,wherein the sequencing process comprises a single molecule sequencingmethod.
 16. The method of claim 1, wherein the sample nucleic acid iscell-free DNA.
 17. The method of claim 1, wherein the sample nucleicacid is obtained from a pregnant female subject.
 18. The method of claim17, wherein the subject is human.
 19. The method of claim 1, wherein thesample nucleic acid is from plasma or serum.